Repressible sterility of animals

ABSTRACT

A construct which allows animals to be bred in captivity but renders them infertile in the wild by allowing reversible control over fertility and reproduction. The construct comprises: a first promoter that is activated in a defined spatial (tissue specific) or temporal manner linked to DNA encoding a transactivating protein; and a second promoter, which is activated by the transacting protein, linked to DNA encoding a blocker molecule which disrupts gametogenesis or embryogenesis. Feeding an animal a molecule that prevents the transactivating protein binding the second promoter controls fertility.

FIELD OF THE INVENTION

This application is concerned with the control of animal reproduction,and especially with preventing the spread of feral and/or geneticallymodified animals. In particular, the present invention relates toconstructs and methods that allow animals to be bred in captivity, butrenders them infertile in the wild, by allowing reversible control overfertility and reproduction.

BACKGROUND OF THE INVENTION

Feral animals are one of the world's major environmental problems.Goats, cats, rabbits and carp are only the more prominent of hundreds ofspecies traded internationally for recreation or agriculture that haveescaped into the wild and formed destructive populations. Terrestrial,freshwater and marine ecosystems are all conspicuously degraded by thesespecies, to the extent that public concern over feral animals has becomea major issue for industries seeking to introduce new species in orderto compete on world markets.

A good recent example is the Pacific oyster. Despite the promise of newjobs in coastal communities and an industry that is worth $50-75 millionannually, recent applications to expand the geographic area for Pacificoyster mariculture facilities in Australia and the United States havebeen rejected indefinitely until the problem of feral oysters can beovercome. Even plans to expand the size of the industry in areas wherefarming already occurs are being blocked for the same reason, followingvery public and often acrimonious debate between industry andconservation-minded elements of the community. Attempts to solve theproblem using current techniques such as triploidy and sterile hybridshave not been successful. Neither technique can guarantee a zero risk ofproducing feral populations, and both also suffer major technicaldifficulties. In the case of oysters, for example, animals sterilisedvia chemical or genetic manipulation of ploidy do not producesignificant amounts of roe, which substantially reduces their marketvalue. Moreover, these animals still produce a small number of viablegametes. So the debate continues to focus on whether degraded beachesare an acceptable price for new industries and jobs.

Hundreds of species of exotic animals are shipped internationally eachday, mainly for recreational purposes. Inevitably, either accidentallyand/or through intentional release, some animals will escape, andestablish feral populations. Sterilisation prior to importation of suchexotics would prevent the establishment of feral populations and removethe risk of forming new problem pest species. A generic means ofsterilisation that prevents development of these feral populations wouldhave huge economic and environmental benefits.

More recently, the containment of genetically modified animals hascaused concern. For example, Salmon containing genes for enhancedproduction of growth hormones have been produced in Europe, New Zealandand North America. Concern has been expressed about the impact of thesefish as “super-competitors”, should they escape and form feralpopulations. Similar concerns have been expressed about other geneticimprovements that deliberately or accidentally enhance competitiveness.This concern has now grown to a point where there is pressure to bansuch modified organisms in toto. However, given their economicsignificance, it may be preferable to have effective biological controlsin place which enable these organisms to be contained within a specificlocality. A sterile feral construct inserted into the geneticallyenhanced stock would prevent development of viable feral populations, aswell as preventing integration of enhanced genes into populations ofwild con-specifics.

Accordingly, some of the major benefits that a sterile feral constructwould offer include:

-   1. Provision of a fail-safe system for preventing the establishment    of feral populations of exotic species. This could fundamentally    change the risk of importing these species, and would reduce public    antagonism to farming of those that have the potential to be    environmentally destructive.-   2. Protection of investments in breeding stocks, for example those    developed by extensive selective breeding programs. Currently, the    commercial advantages from improved stock can be lost when live,    reproductively capable animals are marketed (eg oysters, prawns, and    sheep). Repressible sterility can be used as a “lock and key”    process whereby improved stock could only breed when provided the    correct combination of repressers (and optionally inducers) in    exactly the right sequence.-   3. Production of animals for intentional release that are guaranteed    to be sterile. Release of such sterile animals has been used as a    control mechanism for certain highly fecund pest species, eg.    insects. Repressible sterility technology makes it possible to apply    similar approaches to other, existing pest species, for which there    are currently no “sterile male” equivalents.-   4. Provision of an effective containment mechanism for genetically    modified organisms. Repressible sterility provides just such a    security system for future applications of molecular engineering in    animal production, yet enables safe propagation of these individuals    using conventional rearing facilities. Linking a genetically    engineered process (faster growth, longer spawning seasons, etc.) to    a repressible sterility construct ensures that genetic enhancements    of exotic or native species do not enter wild populations.

One method of containing genetically-modified organisms, namely, plants,is the so-called “terminator gene” or Technology Protection System(TPS). This approach was developed by Delta and Pine Land Company(D&PL), who jointly owns the rights for this invention with USDA-ARS, asdisclosed in U.S. Pat. No. 5,723,765, which is incorporated herein byreference. Essentially, the method stops the seeds of certain plantsfrom germinating, and utilizes:

-   1. A transiently-active promoter operably linked to a first (toxic,    hence lethal) gene, but separated by a blocking sequence which    prevents the lethal gene expression;-   2. A second gene, encoding a recombinase which, upon expression,    excises the blocker sequence; and-   3. A third gene, encoding a tetracycline-controllable blocker of the    recombinase.

Unless the seeds of the plants are transformed with all three genes, andreceive the tetracycline at a precise point, the recombinase isexpressed, resulting in the blocker sequence being excised, and thetoxic gene being expressed.

While this method may function well in plants, it would not function inmany animal species. Few recombinases have been identified that willfunction in animals (and vertebrates in particular) and those that havebeen identified (eg., Cre and Flp recombinase) function in only alimited number of species. Moreover, the use of a toxic substance inanimals may be unacceptable, particularly for those likely to beconsumed. Further, the system requires a number of complex steps, whichare not readily achieved, and once the blocker sequence has been excisedit is virtually impossible to reverse the control process.

Accordingly, there is still a need to provide methods of preventing theescape of exotic and/or genetically modified animals.

We have now developed such a method. We have designed certain geneticconstructs that allow animals to be bred in captivity, but render themreproductively non-viable or infertile in the wild. Moreover, theseconstructs provide reversible control over fertility and reproduction,and are applicable to a wide variety of animal species.

SUMMARY OF THE INVENTION

In its most general aspect, the invention disclosed herein provides anucleic acid construct which may be inserted into the genome of anytarget organism. The construct can use any promoter/gene combinations,provided that they satisfy the criteria of being activated only duringembryonic development and/or gametogenesis, and being crucial forcompletion of embryogenic development and/or gametogenesis.

One type of construct, which is designed to function in a variety oftarget species, comprises:

-   -   a) a native promoter of a crucial gene;    -   b) a blocking DNA sequence (blocker) contoured for and designed        to abrogate the crucial gene's function or to cause its        mis-expression; and    -   c) a genetic switch to regulate controlled expression/repression        of the blocker/gene knockout.

In captivity, expression of the blocker can be repressed in the presenceof a trigger molecule, supplied via the diet or in soluble form, so thatfertilisation occurs and embryos complete development. In the wild,where the trigger molecule is unavailable, the blocker remains activeand the critical gene is disrupted, leading to early death of invasiveprogeny.

Accordingly, in a first aspect, the present invention provides aconstruct for disrupting gametogenesis or embryogenesis in animals,comprising:

-   -   a) a first nucleic acid molecule, which is activated in a        defined spatio-temporal pattern, and which is operably linked to    -   b) a second nucleic acid molecule, which encodes a        transactivating protein; and    -   c) a third nucleic acid molecule, which is operably linked to a        fourth nucleic acid molecule, wherein activation of said first        nucleic acid molecule controls the expression of the second        nucleic acid molecule, which in turn activates the third nucleic        acid molecule, which effects the expression of the fourth        nucleic acid molecule which encodes a blocker molecule which        disrupts gametogenesis or embryogenesis in the animal. Either or        both the first and fourth nucleic acid molecules are transiently        activated or transiently affect development in a defined        spatio-temporal pattern.

Each of the first, second, third and fourth nucleic acids may be genomicDNA, cDNA, RNA, or a hybrid molecule thereof. It will be clearlyunderstood that the term nucleic acid molecule encompasses a full-lengthmolecule, or a biologically active fragment thereof.

Preferably the first nucleic acid molecule is a DNA molecule encoding apromoter region. More preferably the promoter is activated only duringembryonic development and/or gametogenesis, and is crucial forcompletion of embryogenic development and/or gametogenesis. Mostpreferably this DNA molecule has the nucleotide sequence shown in SEQ IDNO:1, SEQ. ID NO:8 SEQ ID NO:60. A sample of SEQ ID NO.1 DNA wasdeposited at the Australian Government Analytical Laboratories on 22Dec. 1999, and accorded the accession number MM99/09098. A sample of SEQID NO.8 DNA was deposited at the Australian Government AnalyticalLaboratories on ______, and accorded the accession number ______. Asample of SEQ ID NO.60 DNA was deposited at the Australian GovernmentAnalytical Laboratories on 23 Dec. 1999, and accorded the accessionnumber NM99/09106.

Preferably the second nucleic acid molecule is a cDNA molecule encodingthe tetracycline-responsive transcriptional activator protein (tTA), asdefined herein, having a nucleotide sequence of SEQ ID NO:2. A sample ofSEQ ID NO.2 cDNA was deposited at the Australian Government AnalyticalLaboratories on 22 Dec. 1999, and accorded the accession numberMM99/09099.

Preferably the third nucleic acid molecule is DNA molecule encoding arepressible promoter. More preferably the promoter consists of the tetresponsive element (TRE) which is coupled to and tightly regulates aminimal promoter region. Most preferably this comprises the tetresponsive element (TRE) and the P_(minCMV) as shown in SEQ ID NO:3. Asample of SEQ ID NO.3 DNA was deposited at the Australian GovernmentAnalytical Laboratories on 22 Dec. 1999, and accorded the accessionnumber MM99/09100.

Preferably the fourth nucleic acid molecule encodes a blocker moleculeselected from the group consisting of antisense RNA, double-stranded RNA(dsRNA), sense RNA and ribozyme. More preferably the molecule is dsRNAor sense RNA that when mis-expressed disrupts development in a definedspatio-temporal pattern. Most preferably this RNA molecule is encoded bythe nucleotide sequence shown in SEQ ID NO:13, SEQ ID NO:62, SEQ IDNO:23, SEQ ID NO:24, and SEQ ID:61. A sample of SEQ ID NO.13 DNA wasdeposited at the Australian Government Analytical Laboratories on 22Dec. 1999, and accorded the accession number MM99/09100. A sample of SEQID NO:62 DNA was deposited at the Australian Government AnalyticalLaboratories on ______, and accorded the accession number ______. Asample of SEQ ID NO.23 DNA was deposited at the Australian GovernmentAnalytical Laboratories on 22 Dec. 1999, and accorded the accessionnumber NM99/09101. A sample of SEQ ID NO.24 DNA was deposited at theAustralian Government Analytical Laboratories on 22 Dec. 1999, andaccorded the accession number NM99/09102. A sample of SEQ ID NO.61 DNAwas deposited at the Australian Government Analytical Laboratories on 23Dec. 1999, and accorded the accession number NM99/09107.

In a second aspect, the present invention provides a nucleic acidmolecule, which encodes a promoter and is transiently activated in adefined spatio-temporal pattern. More preferably, the promoter is activeonly during a narrow window during embryogenesis or larval development.Most preferably the nucleic acid is a promoter having a nucleotidesequence as shown in SEQ ID NO:1, SEQ ID NO:8 and SEQ ID NO:60.

In a third aspect, the present invention provides a nucleic acidmolecule, which encodes a promoter having:

-   -   a) a nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:8        and SEQ ID NO:60; or    -   b) a biologically active fragment of the sequence in a); or    -   c) a nucleic acid molecule which has at least 75% sequence        homology to the sequence in a) or b); or    -   d) a nucleic acid molecule which is capable of hybridizing to        the sequence in a) or b) under stringent conditions.

In a fourth aspect, the present invention provides a nucleic acidmolecule that encodes the coding region of a gene including:

-   -   a) a nucleotide sequence selected from the group consisting of        SEQ ID NO:63, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO 61 or    -   b) a biologically active fragment of any one of the sequences in        a); or    -   c) a nucleic acid molecule which has at least 75% sequence        homology with any one of the sequences disclosed in a) or b); or    -   d) a nucleic acid molecule that is capable of binding to any one        of the sequences disclosed in a) or b) under stringent        conditions.

A sample of SEQ ID NO.63 DNA was deposited at the Australian GovernmentAnalytical Laboratories on 22 Dec. 1999, and accorded the accessionnumber MM99/09100. A sample of SEQ ID NO.23 DNA was deposited at theAustralian Government Analytical Laboratories on 22 Dec. 1999, andaccorded the accession number NM99/09101. A sample of SEQ ID NO.24 DNAwas deposited at the Australian Government Analytical Laboratories on 22Dec. 1999, and accorded the accession number NM99/09102. A sample of SEQID NO.61 DNA was deposited at the Australian Government AnalyticalLaboratories on 23 Dec. 1999, and accorded the accession numberNM99/09107.

In a fifth aspect, the present invention provides a nucleic acidmolecule which encodes a blocker molecule wherein the blocker moleculeis capable of disrupting gametogenesis or embryogenesis in an animal.

Preferably the blocker molecule is selected from the group consisting ofantisense RNA, dsRNA, sense RNA and ribozyme. More preferably themolecule is dsRNA or sense RNA that when mis-expressed disruptsdevelopment in a defined spatio-temporal pattern. Most preferably theblocker molecule is encoded, or partially encoded, by a sequenceselected from the group consisting of SEQ ID NO:13, SEQ ID NO:62, SEQ IDNO:23 and SEQ ID NO:61. A sample of SEQ ID NO.13 DNA was deposited atthe Australian Government Analytical Laboratories on 22 Dec. 1999, andaccorded the accession number MM99/09100. A sample of SEQ. ID NO.62 DNAwas deposited at the Australian Government Analytical Laboratories on______, and accorded the accession number ______. A sample of SEQ IDNO.61 DNA was deposited at the Australian Government AnalyticalLaboratories on 23 Dec. 1999, and accorded the accession numberNM99/09107.

In an sixth aspect, the present invention provides a construct fordisrupting gametogenesis or embryogenesis in animals, comprising:

-   -   a) a first nucleic acid molecule, which is transiently activated        in a defined spatio-temporal pattern, and which is operably        linked to    -   b) a second nucleic acid molecule, which encodes a blocker        molecule.        wherein activation of said first nucleic acid molecule controls        the expression of the second nucleic acid which disrupts        gametogenesis or embryogenesis in the animal.

In a seventh aspect, the present invention provides a method ofpreventing embryogenesis in animals comprising the steps of:

-   -   1) stably transforming an animal cell with a construct according        to the invention; and    -   2) implanting the cell into a host organism, whereby a whole        animal develops from the implanted cell.

Preferably, the stable transformation is effected by microinjection,transfection or infection, wherein the construct stably integrates intothe genome by homologous recombination.

In an eighth aspect, the present invention provides a transgenic animalstably transformed with a construct according to the invention.

Preferably the host organism is of the same genus as the transformedcell. More preferably the host organism is any animal, includingvertebrates and invertebrates. Most preferably the host organism isselected from the group consisting of fish, mammals, amphibians, andmollusc. Fish include; but are not limited to, zebrafish, European carp,salmon, tilapia and trout. Mammals include; but are, not limited to,cats, dogs, donkeys, camels, rabbits, rats, and mice. Molluscs include;but are not limited to, Pacific oysters, zebra mussels, striped mussels,abalone, pearl oysters, and scallops.

Modified and variant forms of the constructs may be produced in vitro,by means of chemical or enzymatic treatment, or in vivo by means ofrecombinant DNA technology. Such constructs may differ from thosedisclosed, for example, by virtue of one or more nucleotidesubstitutions, deletions or insertions, but substantially retain abiological activity of the construct or nucleic acid molecule of thisinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the plasmid map of pBAC5/H11.

FIG. 2 shows the plasmid map of pZBMP2(1.4)-EGFP. The transcriptionalunit consists of the modified EGFP coding sequences (Cormac et al.,1996), under the regulation of a 1,414 bp zBMP2 promoter.

FIG. 3 shows zBMP2 promoter-driven EGFP expression in zebrafish embryoat 9.5 h pi. Right, latero-ventral view, anterior to right. Panel Ashows a typical zebrafish embryo showing EGFP expression predominantlyin the anterio-ventral region. Panel B shows a light micrograph of theembryo on left. PO, polster.

FIG. 4 shows EGFP expression in 9.5 hpi old zebrafish embryo. Lateralviews, with dorsal to top and anterior to left. Panel A shows EGFPexpression driven by zBMP2 promoter. Panel B shows a light micrograph ofthe embryo on left. PO, polster; TB, tail bud.

FIG. 5 shows anterior region of a zebrafish embryo, showing EGFPexpression driven by zpBMP2 at 24-h pi. Panel A shows the left,dorso-lateral view. EGFP expression is seen in domains of native zBMP2expression. Panel B shows light micrograph of the embryo on left. Left,lateral view. PE, posterior margin of eye; OV, otic vesicle; FB,pectoral fin bud.

FIG. 6 shows the plasmid map of pSMAD5-EGFP. A sample of pSMAD5-EGFP wasdeposited at the Australian Government Analytical Laboratories on______, and accorded the accession number ______. The zebrafish smad5promoter drives expression of the EGFP.

FIG. 7 shows a shield stage zebrafish embryo, showing ubiquitousexpression of EGFP (panel A) driven by zebrafish smad5 promoter Panel Brepresents the light micrograph of the embryo on left.

FIG. 8 shows middle section of a typical 24 hpi zebrafish embryoinjected with pSMAd5-EGFP. The EGFP expression is predominantlyrestricted to ventral tissues. D, dorsal; V, ventral.

FIG. 9 shows dorsalized phenotypes of zebrafish, resulting from zBMP2antisense (A) and dsRNA (B) injections. Developments of ventralstructures are perturbed in both instances.

FIG. 10 shows the ventralized chordino phenotypes of zebrafish resultingfrom zBMP2 sense transcript injections. Enlarged blood island (A and B,arrow) and multiplicated ventral margin of tail fin (C, arrow).

FIG. 11 shows the plasmid map of the antisense EGFP fusion construct,pzBMP2-As-EGFP. A sample of pzBMP2-As-EGFP was deposited at theAustralian Government Analytical Laboratories on 22 Dec. 1999, andaccorded the accession number MM99/09102.

FIG. 12 shows the plasmid map of pzBMP2-dsRNA. The zBMP2 promoter drivesthe expression of about 800 bp of zBMP2 cDNA, designed to fold back onitself as a dsRNA.

FIG. 13 shows the plasmid map of pzBMP2-Tet-Off. This construct wasengineered to drive expression of tTA under the regulation of zBMP2promoter.

FIG. 14 shows the plasmid map of the complete sterile feral construct,pSF1. The zBMP2 promoter drives the expression of tTA, which in turnactivates the expression of EGFP and the zBMP2 double stranded RNAblocker, in the absence of doxycycline.

FIG. 15 shows a plasmid map of zebrafish Sterile feral Construct pSF2.This construct is identical to pSF1, except that CMV promoter drive'sthe tTA. A sample of pSF2 was deposited at the Australian GovernmentAnalytical Laboratories on ______, and accorded the accession number______.

FIG. 16 shows a plasmid map of zebrafish Sterile feral Construct pSF3.This construct is identical to pSF2, except that the zebrafish smad5promoter drives the tTA. A sample of pSF3 was deposited at theAustralian Government Analytical Laboratories on ______, and accordedthe accession number ______.

FIG. 17 shows a plasmid map of zebrafish Sterile feral Construct pSF4.This construct is identical to pSF3, except that the zBMP2 doublestranded RNA blocker is replaced by zBMP2 sense cDNA. A sample of pSF4was deposited at the Australian Government Analytical Laboratories on______, and accorded the accession number

FIG. 18 (A-C) show 24-hpi zebrafish embryos following the injection ofpSF4. Panel A, two-zebrafish embryos with enlarged blood islands(arrow), typical of ventralized mutations. Panel B, close up view of 24hpi zebrafish embryo tail, with enlarged blood island (arrow). Panel C,EGFP micrograph of embryo in panel B, showing close association of EGFPexpression and ventralization (arrow).

FIG. 19 shows the amino acid alignments of closely related HOXCG1 andHOXCG3 genes in various animals.

FIG. 20 shows (a) typical control D-hinge larvae with a single velum and(b) a larvae exhibiting the multiple velum phenotype as a consequence ofblocking expression of Hox CG1 with double stranded HOXG1 RNA.

FIG. 21 shows the plasmid map of the double stranded blocking constructfor oyster Hox gene, pBiT(dHSP)-RFP-oHoxDS/BH. A sample ofpBiT(dHSP)-RFP-oHoxDS/BH was deposited at the Australian GovernmentAnalytical Laboratories on ______, and accorded the accession number______.

FIG. 22 shows the amino acid alignments of closely related goosecoidgenes in various animals.

FIG. 23 shows the mechanisms of action of regulatory elements of themouse goosecoid gene promoter region.

FIG. 24 shows the plasmid map of the mouse goosecoid promoter drivingexpression of the enhanced green fluorescent protein reporter (pSFM 1).

FIG. 25 shows the plasmid map of the tetracycline transactivated TREdriving expression of the mouse goosecoid cDNA (pSFM 2).

FIG. 26 shows the mouse goosecoid promoter driving expression of mousegoosecoid cDNA fused to the red fluorescent protein reporter (pSFM 6).

FIG. 27 shows the plasmid map of the mouse goosecoid promoter drivingexpression of the tetracycline transactivator tTA protein (pSFM 7).

FIG. 28 shows the plasmid map of the mouse goosecoid promoter drivingexpression of the luciferase+ protein reporter (pSFM 20).

FIG. 29 shows the plasmid map of the promoter-less luciferase+ proteinreporter (pSFM 21).

FIG. 30 shows the plasmid map of the CMV promoter driving expression ofthe luciferase+ protein reporter (pSFM 23).

FIG. 31 shows the plasmid map of the tetracycline transactivated TREdriving expression of the enhanced green fluorescent protein reporter(pSFM 24).

FIG. 32 shows the plasmid map of the tetracycline transactivated TREdriving expression of the luciferase+ protein reporter (pSFM 25).

FIG. 33 shows an agarose gel demonstrating the presence of mousegoosecoid mRNA expression in P19 cells as detected by RT-PCRamplification of mRNA using goosecoid-specific primers. Lane 1: PCRproduct from P19 cells using goosecoid primers; Lane 2: PCR product from1 fg of pSFM 2 as a positive goosecoid control; Lane 3: PCR product fromP19 cells with GAPDH primers; Lane 4: DNA MW marker.

FIG. 34 shows the plasmid map of the tetracycline transactivated TREdriving expression of the mouse goosecoid dsRNA blocker construct (pSFM5).

FIG. 35 shows the plasmid map of the CMV promoter driving expression ofthe mouse goosecoid antisense RNA blocker construct (pSFM 8).

FIG. 36 shows the plasmid map of the tetracycline transactivated TREdriving expression of the mouse goosecoid antisense blocker construct(pSFM 9). A sample of pSFM 9 was deposited at the Australian GovernmentAnalytical Laboratories on 23 Dec. 1999 and accorded the accessionnumber NM99/09107.

FIG. 37 shows the cellular locations of CMV promoter-driven expressionof red fluorescent protein in P19-SFM 7 cells (A,B), CMV promoter-drivenexpression of red fluorescent protein fused to the mouse goosecoidprotein (C) and TRE tetracycline responsive enhanced green fluorescentprotein expression in cells co-transfected with CMV promoter-drivenexpression of red fluorescent protein fused to the mouse goosecoidprotein (D).

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention employs, unless otherwiseindicated, conventional molecular biology, microbiology, and recombinantDNA techniques within the skill of the art. Such techniques are wellknown to the skilled worker, and are explained fully in the literature.See, e.g., “DNA Cloning: A Practical Approach,” Volumes I and II (D. N.Glover, ed., 1985); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984);“Nucleic Acid Hybridization” (B. D. Hames & S. J. Higgins, eds., 1985);“Transcription and Translation” (B. D. Hames & S. J. Higgins, eds.,1984); “Animal Cell Culture” (R. I. Freshney, ed., 1986); “ImmobilizedCells and Enzymes” (IRL Press, 1986); B. Perbal, “A Practical Guide toMolecular Cloning” (1984), and Sambrook, et al., “Molecular Cloning: aLaboratory Manual” 12^(th) edition (1989).

Definitions

The description that follows makes use of a number of terms used inrecombinant DNA technology. In order to provide a clear and consistentunderstanding of the specification and claims, including the scope givensuch terms, the following definitions are provided.

A “nucleic acid molecule” or “polynucleic acid molecule” refers hereinto deoxyribonucleic acid and ribonucleic acid in all their forms, i.e.,single and double-stranded DNA, cDNA, mRNA, and the like.

A “double-stranded DNA molecule” refers to the polymeric form ofdeoxyribonucleotides (adenine, guanine, thymine, or cytosine) in itsnormal, double-stranded helix. This term refers only to the primary andsecondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the non-transcribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

A DNA sequence “corresponds” to an amino acid sequence if translation ofthe DNA sequence in accordance with the genetic code yields the aminoacid sequence (i.e., the DNA sequence “encodes” the amino acidsequence).

One DNA sequence “corresponds” to another DNA sequence if the twosequences encode the same amino acid sequence.

Two DNA sequences are “substantially similar” when at least about 85%,preferably at least about 90%, and most preferably at least about 95%,of the nucleotides match over the defined length of the DNA sequences.Sequences that are substantially similar can be identified in a Southernhybridization experiment, for example under stringent conditions asdefined for that particular system. Defining appropriate hybridizationconditions is within the skill of the art. See e.g., Sambrook et al.,“Molecular Cloning: a Laboratory Manual” 12^(th) edition (1989), vols.I, II and III. Nucleic Acid Hybridization. However, ordinarily,“stringent conditions” for hybridization or annealing of nucleic acidmolecules are those that (1) employ low ionic strength and hightemperature for washing, for example, 0.015 M NaCl/0.0015 M sodiumcitrate/0.1% sodium dodecyl sulfate (SDS) at 50° C., or (2) employduring hybridization a denaturing agent such as formamide, for example,50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42° C.

Another example is use of 50% formamide, 5×SSC (0.75 M NaCl, 0.075 Msodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5× “Denhardt's solution, sonicated salmon sperm DNA (50μg/mL), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42°C. in 0.2×SSC and 0.1% SDS.

A “heterologous” region or domain of a DNA construct is an identifiablesegment of DNA within a larger DNA molecule that is not found inassociation with the larger molecule in nature. Thus, when theheterologous region encodes a mammalian gene, the gene will usually beflanked by DNA that does not flank the mammalian genomic DNA in thegenome of the source organism. Another example of a heterologous regionis a construct where the coding sequence itself is not found in nature(e.g., a cDNA where the genomic coding sequence contains introns, orsynthetic sequences having codons different than the native gene).Allelic variations or naturally occurring mutational events do not giverise to a heterologous region of DNA as defined herein.

A “gene” includes all the DNA sequences associated with the promoter andcoding region and non-coding region such as introns and 5′ and 3′non-coding sequences and enhancer elements.

A “coding region” is an in-frame sequence of codons from the startcodon, normally ATG, to the stop codon TAA, and which may or may notinclude introns.

A “coding sequence” is an in-frame sequence of codons that correspond toor encode a protein or peptide sequence. Two coding sequences correspondto each other if the sequences or their complementary sequences encodethe same amino acid sequences. A coding sequence in association withappropriate regulatory sequences may be transcribed and translated intoa polypeptide in vivo. A polyadenylation signal and transcriptiontermination sequence will usually be located 3′ to the coding sequence.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream(3′direction) coding sequence. A coding sequence is “under the control”of the promoter sequence in a cell when RNA polymerase which binds thepromoter sequence transcribes the coding sequence into mRNA, which isthen in turn translated into the protein encoded by the coding sequence.

For the purposes of the present invention, the promoter sequence isbounded at its 3′ terminus by the translation start codon of a codingsequence, and extends upstream to include the minimum number of bases orelements necessary to initiate transcription at levels detectable abovebackground. Within the promoter sequence will be found a transcriptioninitiation site (conveniently defined by mapping with nuclease S1), aswell as protein binding domains (consensus sequences) responsible forthe binding of RNA polymerase. Eukaryotic promoters will often, but notalways, contain “TATA” boxes and “CAT” boxes, prokaryotic promoterscontain Shine-Delgarno sequences in addition to the −10 and −35consensus sequences.

A cell has been “transformed” by exogenous DNA when such exogenous DNAhas been introduced inside the cell wall. Exogenous DNA may or may notbe integrated (covalently linked) to chromosomal DNA making up thegenome of the cell. In prokaryotes and yeast, for example, the exogenousDNA may be maintained on an episomal element such as a plasmid. Withrespect to eukaryotic cells, a stably transformed cell is one in whichthe exogenous DNA is inherited by daughter cells through chromosomereplication. This stability is demonstrated by the ability of theeukaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the exogenous DNA.

“Integration” of the DNA may be effected using non-homologousrecombination following mass transfer of DNA into the cells usingmicroinjection, biolistics, electroporation or lipofection. Alternativemethods such as homologous recombination, and or restriction enzymemediated integration (REMI) or transposons are also encompassed, and maybe considered to be improved integration methods.

A “clone” is a population of cells derived from a single cell or commonancestor by mitosis.

“Cell,” “host cell,” “cell line,” and “cell culture” are usedinterchangeably herewith and all such terms should be understood toinclude progeny. A “cell line” is a clone of a primary cell that iscapable of stable growth in vitro for many generations. Thus the words“transformants” and “transformed cells” include the primary subject celland cultures derived therefrom, without regard for the number of timesthe cultures have been passaged. It should also be understood that allprogeny might not be precisely identical in DNA content, due todeliberate or inadvertent mutations.

Vectors are used to introduce a foreign substance, such as DNA, RNA orprotein, into an organism. Typical vectors include recombinant viruses(for DNA) and liposomes (for protein). A “DNA cloning vector” is anautonomously replicating DNA molecule,” such as plasmid, phage orcosmid. Typically the DNA cloning vector comprises one or a small numberof restriction endonuclease recognition sites, at which such DNAsequences may be cut in a determinable fashion without loss of anessential biological function of the vector, and into which a DNAfragment may be spliced in order to bring about its replication andcloning. The cloning vector may also comprise a marker suitable for usein the identification of cells transformed with the cloning vector.

An “expression vector” is similar to a DNA cloning vector, but containsregulatory sequences which are able to direct protein synthesis by anappropriate host cell. This usually means a promoter to bind RNApolymerase and initiate transcription of mRNA, as well as ribosomebinding sites and initiation signals to direct translation of the mRNAinto a polypeptide. Incorporation of a DNA sequence into an expressionvector at the proper site and in correct reading frame, followed bytransformation of an appropriate host cell by the vector, enables theproduction of mRNA corresponding to the DNA sequence, and usually of aprotein encoded by the DNA sequence.

“Plasmids” are DNA molecules that are capable of replicating within ahost cell, either extrachromosomally or as part of the host cellchromosome(s), and are designated by a lower case “p” preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare commercially available, are publicly available on an unrestrictedbasis, or can be constructed from such available plasmids by methodsdisclosed herein and/or in accordance with published procedures. Incertain instances, as will be apparent to the ordinarily skilled worker,other plasmids known in the art may be used interchangeably withplasmids described herein.

“Control sequences” refers to DNA sequences necessary for the expressionof an operably linked nucleotide coding sequence in a particular hostcell. The control sequences suitable for expression in prokaryotes, forexample, include origins of replication, promoters, ribosome bindingsites, and transcription termination sites. The control sequences thatare suitable for expression in eukaryotes, for example, include originsof replication, promoters, ribosome binding sites, polyadenylationsignals, and enhancers.

An “exogenous” element is one that is foreign to the host cell, or ishomologous to the host cell but in a position within the host cell inwhich the element is ordinarily not found.

“Digestion” of DNA refers to the catalytic cleavage of DNA with anenzyme that acts only at certain locations in the DNA. Such enzymes arecalled restriction enzymes or restriction endonucleases, and the siteswithin DNA where such enzymes cleave are called restriction sites. Ifthere are multiple restriction sites within the DNA, digestion willproduce two or more linearized DNA fragments (restriction fragments).The various restriction enzymes used herein are commercially available,and their reaction conditions, cofactors, and other requirements asestablished by the enzyme manufacturers are used. Restriction enzymesare commonly designated by abbreviations composed of a capital letterfollowed by other letters representing the microorganism from which eachrestriction enzyme originally was obtained and then a number designatingthe particular enzyme. In general, about 1 μg of DNA is digested withabout 1-2 units of enzyme in about 20 μl of buffer solution. Appropriatebuffers and substrate amounts for particular restriction enzymes arespecified by the manufacturer, and/or are well known in the art.

“Recovery” or “isolation” of a given fragment of DNA from a restrictiondigest typically is accomplished by separating the digestion products,which are referred to as “restriction fragments,” on a polyacrylamide oragarose gel by electrophoresis, identifying the fragment of interest onthe basis of its mobility relative to that of marker DNA fragments ofknown molecular weight, excising the portion of the gel that containsthe desired fragment, and separating the DNA from the gel, for exampleby electroelution.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double-stranded DNA fragments. Unless otherwise specified, ligationis accomplished using known buffers and conditions with 10 units of T4DNA ligase per 0.5 μg of approximately equimolar amounts of the DNAfragments to be ligated.

“Oligonucleotides” are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods(involving, for example, triester, phosphoramidite, or phosphonatechemistry), such as described by Engels et al., Agnew. Chem. Int. Ed.Engl. 28:716-734 (1989). They are then purified, for example, bypolyacrylamide gel electrophoresis.

“Polymerase chain reaction,” or “PCR,” as used herein generally refersto a method for amplification of a desired nucleotide sequence in vitro,as described in U.S. Pat. No. 4,683,195. In general, the PCR methodinvolves repeated cycles of primer extension synthesis, using twooligonucleotide primers capable of hybridizing preferentially to atemplate nucleic acid. Typically, the primers used in the PCR methodwill be complementary to nucleotide sequences within the template atboth ends of or flanking the nucleotide sequence to be amplified,although primers complementary to the nucleotide sequence to beamplified also may be used. See Wang et al., in PCR Protocols, pp.70-75(Academic Press, 1990); Ochman et al., in PCR Protocols, pp. 219-227;Triglia, et al., Nuc. Acids Res. 16:8186 (1988).

“PCR cloning” refers to the use of the PCR method to amplify a specificdesired nucleotide sequence that is present amongst the nucleic acidsfrom a suitable cell or tissue source, including total genomic DNA andcDNA transcribed from total cellular RNA. See Frohman et al., Proc. Nat.Acad. Sci. USA 85:8998-9002 (1988); Saiki et al., Science. 239:487-492(1988); Mullis et al., Meth. Enzymol. 155:335-350 (1987).

“zBMP2 promoter” refers to a promoter encoded by the nucleotide sequenceset forth in SEQ ID NO.:1. “zSMAD promoter” refers to a promoter encodedby the nucleotide sequence set forth in SEQ ID NO.:8. “goosecoidpromoter” refers to a promoter encoded by the nucleotide sequence setforth in SEQ ID NO:60. “Blocker molecule” refers to either antisenseRNA, dsRNA, sense RNA or DNA that preferably encodes BMP2, GSC, HoxCG1or HoxCG3 and includes the sequences shown in SEQ ID NO:13, SEQ IDNO:20, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:61. However, it will beappreciated by those skilled in the art that any nucleic acid moleculecapable of disrupting gametogenesis or embryogenesis is encompassed.Accordingly, the terms “blocker molecule RNA” and “blocker molecule DNA”as used herein are interchangeable depending upon whether it is aspecies of RNA or DNA, that is being addressed. “HoxCG” refers to genesHoxCG1 and HoxCG3 isolated from Pacific oyster encoded by the nucleotidesequences set forth in SEQ ID NO.:23 and SEQ ID NO:24, respectively.Sequence variants of zBMP2 promoter, SMAD promoter, goosecoid promoterand HoxCG blocker molecules may be made synthetically, for example, bysite-directed or PCR mutagenesis, or may exist naturally, as in the caseof allelic forms and other naturally occurring variants of thenucleotide sequences set forth in SEQ ID NO.:1, SEQ ID NO:8, SEQ IDNO:60, SEQ ID NO:23, and SEQ ID NO:24, respectively, that may occur infish and other animal species.

zBMP2 promoter, SMAD promoter, goosecoid promoter HoxCG, and blockermolecule nucleotide sequence variants are included within the scope ofthe invention, provided that they are functionally active. As usedherein, “functionally active” and “functional activity” with referenceto zBMP2 promoter, SMAD promoter, goosecoid promoter and HoxCG meansthat the zBMP2 promoter, SMAD promoter, goosecoid promoter and HoxCGvariants are able to function in a similar way to naturally occurringzBMP2 promoter, SMAD promoter, goosecoid promoter and HoxCG. Withreference to the blocker molecule “functionally active” and “functionalactivity” means that the blocker molecule variants are capable ofdisrupting gametogenesis or embyrogenesis in an animal. Therefore, zBMP2promoter, SMAD promoter, goosecoid promoter HoxCG and blocker moleculenucleotide sequence variants generally will share at least about 75%,preferably greater than 80% and more preferably greater than 90%,sequence identity with the nucleotide sequences set forth in SEQ IDNO.:1, SEQ ID NO:8, SEQ ID NO:60, SEQ ID NO:23, and SEQ ID NO:24respectively, after aligning the sequences to provide for maximumhomology, as determined, for example, by the Fitch et al., Proc. Nat.Acad. Sci. USA 80:1382-1386 (1983), version of the algorithm describedby Needleman et al., J. Mol. Biol. 48:443-453 (1970).

Nucleotide sequence variants of zBMP2 promoter, SMAD promoter, goosecoidpromoter HoxCG and blocker molecule are prepared by introducingappropriate nucleotide changes into zBMP2 promoter, SMAD promoter,goosecoid promoter, HoxCG and blocker molecule DNA, or by in vitrosynthesis. Such variants include deletions from, or insertions orsubstitutions of, nucleotides within the zBMP2 promoter, SMAD promoter,goosecoid promoter, HoxCG or blocker molecule nucleotide sequences setforth in SEQ ID NO.:1, SEQ ID NO:8, SEQ ID NO: 60, SEQ ID NO:23, and SEQID NO:24. Any combination of deletion, insertion, and substitution maybe made to arrive at a nucleotide sequence variant of zBMP2 promoter,SMAD promoter, goosecoid promoter HoxCG or blocker molecule providedthat such variants possess the desired characteristics described herein.Changes that are made in the nucleotide sequence set forth in SEQ IDNO.:1, SEQ ID NO:8, SEQ ID NO:60, SEQ ID NO:23, and SEQ ID NO:24,respectively, to arrive at nucleotide sequence variants of zBMP2promoter, SMAD promoter, goosecoid promoter and HoxCG blocker moleculesalso may result in further modifications of the zBMP2 promoter, SMADpromoter, goosecoid promoter, HoxCG or blocker molecule upon theiractivation in host cells.

There are two principal variables in the construction of nucleotidesequence variants of zBMP2 promoter, SMAD promoter, goosecoid promoter,HoxCG and blocker molecule nucleic acid: the location of the mutationsite and the nature of the mutation. These are variants from thenucleotide sequences set forth in SEQ ID NO.:1, SEQ ID NO:8, SEQ ID NO60, SEQ ID NO:23, and SEQ ID NO:24 and may represent naturally occurringallelic forms of zBMP2 promoter, SMAD promoter, goosecoid promoter,HoxCG and blocker molecule or predetermined mutant forms of zBMP2promoter, SMAD promoter, goosecoid promoter, HoxCG and blocker moleculemade by mutating zBMP2 promoter, SMAD promoter, goosecoid promoter,HoxCG or blocker molecule DNA, either to arrive at an allele or avariant not found in nature. In general, the location and nature of themutation chosen will depend upon the zBMP2 promoter, SMAD promoter,goosecoid promoter, HoxCG or blocker molecule characteristic to bemodified.

Nucleotide sequence deletions generally range from about 1 to 30nucleotides, more preferably about 1 to 10 nucleotides, and aretypically contiguous.

Nucleotide sequence insertions include fusions ranging in length fromone nucleotide to hundreds of nucleotides, as well as intrasequenceinsertions of single or multiple nucleotides. Intrasequence insertions(i.e., insertions made within the nucleotide sequences set forth in SEQID NO:1, SEQ ID NO:8, SEQ ID NO:60, SEQ ID NO:23, and SEQ ID NO:24) mayrange generally from about 1 to 10 nucleotides, more preferably 1 to 5,most preferably 1 to 3.

The third group of variants are those in which nucleotides in thenucleotide sequences set forth in SEQ ID NO.:1, SEQ ID NO:8, SEQ IDNO:60, SEQ ID NO.23, and SEQ ID NO:24 have been substituted with othernucleotides. Preferably one to four, more preferably one to three, evenmore preferably one to two, and most preferably only one nucleotide hasbeen removed and a different nucleotide inserted in its place. The sitesof greatest interest for making such substitutions are those sites thatare likely to be important to the functional activity of the zBMP2promoter, SMAD promoter, goosecoid promoter, HoxCG or blocker molecule.

zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG and blockermolecule DNA is obtained from cDNA or genomic DNA libraries, or by invitro synthesis. Identification of zBMP2 promoter, SMAD promoter,goosecoid promoter, HoxCG or blocker molecule DNA within a cDNA or agenomic DNA library, or in some other mixture of various DNAs, isconveniently accomplished by the use of an oligonucleotide hybridizationprobe labelled with a detectable moiety, such as a radioisotope. SeeKeller et al., DNA Probes, pp.149-213 (Stockton Press, 1989). Toidentify DNA encoding zBMP2 promoter, SMAD promoter, goosecoid promoter,HoxCG or blocker molecule DNA, the nucleotide sequence of thehybridization probe is preferably selected so that the hybridizationprobe is capable of hybridizing preferentially to DNA encodinghomologues of the equivalent zBMP2 promoter, SMAD promoter, goosecoidpromoter, HoxCG or blocker molecule DNA in other species, or variants orderivatives thereof as described herein, under the hybridizationconditions chosen. Another method for obtaining zBMP2 promoter, SMADpromoter, goosecoid promoter, HoxCG or blocker molecule is chemicalsynthesis using one of the methods described, for example, by Engels etal., Agnew. Chem. Int. Ed. Engl. 28:716-734 (1989).

If the entire nucleotide coding sequence for zBMP2 promoter, SMADpromoter, goosecoid promoter, HoxCG or blocker molecule is not obtainedin a single cDNA, genomic DNA, or other DNA, as determined, for example,by DNA sequencing or restriction endonuclease analysis, then appropriateDNA fragments (e.g., restriction fragments or PCR amplificationproducts) may be recovered from several DNA's, and covalently joined toone another to construct the entire coding sequence. The preferred meansof covalently joining DNA fragments is by ligation using a DNA ligaseenzyme, such as T4 DNA ligase.

“Isolated” zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG orblocker molecule nucleic acid is zBMP2 promoter, SMAD promoter,goosecoid promoter, HoxCG or blocker molecule nucleic acid that isidentified and separated from (or otherwise substantially free from),contaminant nucleic acid encoding other polypeptides. The isolated zBMP2promoter, SMAD promoter, goosecoid promoter, HoxCG or blocker moleculecan be incorporated into a plasmid or expression vector, or can belabeled for probe purposes, using a label as described further herein inthe discussion of assays and nucleic acid hybridization methods.

It will be appreciated that if the desired result of the presentinvention is sterilized adult feral animals then the blocker moleculesmay be expressed in vitro, isolated, purified, and then delivered tospecific organisms. The mode of delivery may be any known procedureincluding injection and ingestion. Moreover, constructs of the presentinvention which are capable of expressing blocker molecules may also bedelivered to adult feral animals by viral vectors like adenovirus.Isolated zBMP2 promoter, SMAD promoter and goosecoid promoter nucleicacid is also used to control the expression of other desired genes orblocker molecules in vivo. Indeed, the zBMP2 promoter, SMAD promoter andgoosecoid promoter may be used in any vector, or construct where theexpression of a gene, cDNA, or coding sequence is desirably controlledto be at a particular spatio-temporal point during embyrogenesis. Itwill be appreciated that while the zBMP2 promoter and SMAD promoter areparticularly useful in controlling the expression of nucleic acids infish, they are equally useful in other organisms. In various embodimentsof the invention, host cells are transformed or transfected withrecombinant DNA molecules comprising an isolated zBMP2 promoter or SMADpromoter DNA or goosecoid promoter operably linked to a desired nucleicacid molecule, wherein the expression of the desired molecule isdirectly or indirectly under the control of the zBMP2 promoter or SMADpromoter or goosecoid promoter.

Isolated HoxCG nucleic acid is also used to produce HoxCG by recombinantDNA and recombinant cell culture methods. In various embodiments of theinvention, host cells are transformed or transfected with recombinantDNA molecules comprising an isolated HoxCG DNA, to obtain expression ofthe HoxCG DNA and thus the production of HoxCG in large quantities. DNAencoding amino acid sequence variants of HoxCG is prepared by a varietyof methods known in the art. These methods include, but are not limitedto, isolation from a natural source (in the case of naturally occurringamino acid sequence variants of HoxCG), or preparation by site-directedor oligonucleotide-mediated mutagenesis, PCR mutagenesis, and cassettemutagenesis of DNA encoding a variant or a non-variant form of HoxCG.

Site-directed mutagenesis is a preferred method for preparingsubstitution, deletion, and insertion variants of HoxCG DNA, or otherDNA such as the zBMP2 promoter, SMAD promoter, and blocker molecule DNA.This technique is well known in the art; see Zoller et al., Meth. Enz.100:4668-500 (1983); Zoller, et al., Meth. Enz. 154:329-350 (1987);Carter, Meth. Enz. 154:382-403 (1987); Horwitz et al., Meth. Enz.185:599-611 (1990), and has been used to produce amino acid sequencevariants of trypsin and T4 lysozyme, which variants have certain desiredfunctional properties. Perry et al., Science 226:555-557 (1984); Craiket al., Science 228:291-297 (1985).

Briefly, in carrying out site-directed mutagenesis of zBMP2 promoter,SMAD promoter, goosecoid promoter, HoxCG and blocker molecule DNA, thezBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG and blockermolecule DNA is altered by first hybridizing an oligonucleotide encodingthe desired mutation to a single strand of zBMP2 promoter, SMADpromoter, goosecoid promoter, HoxCG and blocker molecule DNA. Afterhybridization, a DNA polymerase is used to synthesize an entire secondstrand, using the hybridized oligonucleotide as a primer, and using thesingle strand of zBMP2 promoter, SMAD promoter, goosecoid promoter,HoxCG and blocker molecule DNA as a template. Thus the oligonucleotideencoding the desired mutation is incorporated into the resultingdouble-stranded DNA.

Oligonucleotides for use as hybridization probes or primers may beprepared by any suitable method, such as purification of a naturallyoccurring DNA or in vitro synthesis. For example, oligonucleotides arereadily synthesized using various techniques in such as those describedby Narang et al., Meth. Enzymol. 68:90-98 (1979); Brown et al., Meth.Enzymol. 68:109-151 (1979); Caruther et al., Meth. Enzymol. 154:287-313(1985). The general approach to selecting a suitable hybridization probeor primer is well known. Keller et al., DNA Probes, pp.11-18 (StocktonPress, 1989). Typically, the hybridization probe or primer will contain10-25 or more nucleotides, and will include at least 5 nucleotides oneither side of the sequence encoding the desired mutation so as toensure that the oligonucleotide will hybridize preferentially to thesingle-stranded DNA template molecule.

Multiple mutations are introduced into HoxCG DNA to produce amino acidsequence variants of HoxCG comprising several or a combination ofinsertions, deletions, or substitutions of amino acid residues ascompared to the amino acid sequences set forth in FIG. 20. If the sitesto be mutated are located close together, the mutations may beintroduced simultaneously using a single oligonucleotide that encodesall of the desired mutations. If, however, the sites to be mutated arelocated some distance from each other (separated by more than about tennucleotides), it is more difficult to generate a single oligonucleotidethat encodes all of the desired changes. Instead, one of two alternativemethods may be employed.

In the first method, a separate oligonucleotide is generated for eachdesired mutation. The oligonucleotides are then simultaneously annealedto the single-stranded template DNA, and the second strand of DNA thatis synthesized from the template will encode all of the desired aminoacid substitutions.

The alternative method involves two or more rounds of mutagenesis toproduce the desired mutant. The first round is as described forintroducing a single mutation: a single strand of a previously preparedHoxCG DNA is used as a template, an oligonucleotide encoding the firstdesired mutation is annealed to this template, and a heteroduplex DNAmolecule is then generated. The second round of mutagenesis utilizes themutated DNA produced in the first round of mutagenesis as the template.Thus this template already contains one or more mutations. Theoligonucleotide encoding the additional desired amino acidsubstitution(s) is then annealed to this template, and the resultingstrand of DNA now encodes mutations from both the first and secondrounds of mutagenesis. This resultant DNA can be used as a template in athird round of mutagenesis, and so on.

PCR mutagenesis is also suitable for making nucleotide sequence variantsof zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG and blockermolecule. Higuchi, in PCR Protocols, pp.177-183 (Academic Press, 1990);Vallette et al., Nuc. Acids Res. 17:723-733 (1989). Briefly, when smallamounts of template DNA are used as starting material in a PCR, primersthat differ slightly in sequence from the corresponding region in atemplate DNA can be used to generate relatively large quantities of aspecific DNA fragment that differs from the template sequence only atthe positions where the primers differ from the template. Forintroduction of a mutation into a plasmid DNA, for example, one of theprimers is designed to overlap the position of the mutation and tocontain the mutation; the sequence of the other primer must be identicalto a nucleotide sequence within the opposite strand of the plasmid DNA,but this sequence can be located anywhere along the plasmid DNA. It ispreferred, however, that the sequence of the second primer is locatedwithin 200 nucleotides from that of the first, such that in the end theentire amplified region of DNA bounded by the primers can be easilysequenced. PCR amplification using a primer pair like the one justdescribed results in a population of DNA fragments that differ at theposition of the mutation specified by the primer, and possibly at otherpositions, as template copying is somewhat error-prone. See Wagner etal., in PCR Topics, pp.69-71 (Springer-Verlag, 1991).

If the ratio of template to product amplified DNA is extremely low, themajority of product DNA fragments incorporate the desired mutation(s).This product DNA is used to replace the corresponding region in theplasmid that served as PCR template using standard recombinant DNAmethods. Mutations at separate positions can be introducedsimultaneously by either using a mutant second primer, or performing asecond PCR with different mutant primers and ligating the two resultingPCR fragments simultaneously to the plasmid fragment in a three (ormore)-part ligation.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al., Gene, 34:315-323 (1985). Thestarting material is the plasmid (or other vector) comprising the zBMP2promoter, SMAD promoter, goosecoid promoter, HoxCG or blocker moleculeDNA to be mutated. The codon(s) in the zBMP2 promoter, SMAD promoter,goosecoid promoter, HoxCG or blocker molecule DNA to be mutated areidentified. There must be a unique restriction endonuclease site on eachside of the identified mutation site(s). If no such restriction sitesexist, they may be generated using the above-describedoligonucleotide-mediated mutagenesis method to introduce them atappropriate locations in the zBMP2 promoter, SMAD promoter, goosecoidpromoter, HoxCG and blocker molecule DNA. The plasmid DNA is cut atthese sites to linearize it. A double-stranded oligonucleotide encodingthe sequence of the DNA between the restriction sites but containing thedesired mutation(s) is synthesized using standard procedures, whereinthe two strands of the oligonucleotide are synthesized separately andthen hybridized together using standard techniques. This double-strandedoligonucleotide is referred to as the cassette. This cassette isdesigned to have 5′ and 3′ ends that are compatible with the ends of thelinearized plasmid, such that it can be directly ligated to the plasmid.This plasmid now contains the mutated zBMP2 promoter, SMAD promoter,goosecoid promoter, HoxCG, or blocker molecule DNA sequence.

zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG, and blockermolecule DNA, whether cDNA or genomic DNA or a product of in vitrosynthesis, is ligated into a replicable vector for further cloning orfor expression. “Vectors” are plasmids and other DNA's that are capableof replicating autonomously within a host cell, and as such, are usefulfor performing two functions in conjunction with compatible host cells(a vector-host system). One function is to facilitate the cloning of thenucleic acid that encodes the zBMP2 promoter, SMAD promoter, goosecoidpromoter, HoxCG, and blocker molecule, i.e., to produce usablequantities of the nucleic acid. The other function is to direct theexpression of HoxCG. One or both of these functions are performed by thevector-host system. The vectors will contain different componentsdepending upon the function they are to perform as well as the host cellwith which they are to be used for cloning or expression.

To produce HoxCG, an expression vector will contain nucleic acid thatencodes HoxCG as described above. The HoxCG of this invention may beexpressed directly in recombinant cell culture, or as a fusion with aheterologous polypeptide, preferably a signal sequence or otherpolypeptide having a specific cleavage site at the junction between theheterologous polypeptide and the HoxCG.

In one example of recombinant host cell expression, cells aretransfected with an expression vector comprising HoxCG DNA and the HoxCGencoded thereby is recovered from the culture medium in which therecombinant host cells are grown. But the expression vectors and methodsdisclosed herein are suitable for use over a wide range of prokaryoticand eukaryotic organisms.

Prokaryotes may be used for the initial cloning of DNA's and theconstruction of the vectors useful in the invention. However,prokaryotes may also be used for expression of mRNA or protein encodedby HoxCG. Polypeptides that are produced inprokaryotic host cellstypically will be non-glycosylated.

Plasmid or viral vectors containing replication origins and othercontrol sequences that are derived from species compatible with the hostcell are used in connection with prokaryotic host cells, for cloning orexpression of an isolated DNA. For example, E. coli typically istransformed using pBR322 a plasmid derived from an E. coli species.Bolivar et al., Gene 2:95-113 (1987). PBR322 contains genes forampicillin and tetracycline resistance so that cells transformed by theplasmid can easily be identified or selected. For it to serve as anexpression vector, the pBR322 plasmid, or other plasmid or viral vector,must also contain, or be modified to contain, a promoter that functionsin the host cell to provide messenger RNA (mRNA) transcripts of a DNAinserted downstream of the promoter. Rangagwala et al., Bio/Technology9:477-479 (1991).

In addition to prokaryotes, eukaryotic microbes, such as yeast, may alsobe used as hosts for the cloning or expression of DNA's useful in theinvention. Saccharomyces cerevisiae, or common baker's yeast, is themost commonly used eukaryotic microorganism. Plasmids useful for cloningor expression in yeast cells of a desired DNA are well known, as arevarious promoters that function in yeast cells to produce mRNAtranscripts.

Furthermore, cells derived from multicellular organisms also may be usedas hosts for the cloning or expression of DNA's useful in the invention.Mammalian cells are most commonly used, and the procedures formaintaining or propagating such cells in vitro, which procedures arecommonly referred to as tissue culture, are well known. Kruse &Patterson, eds., Tissue Culture (Academic Press, 1977). Examples ofuseful mammalian cells are human cell lines such as 293, HeLa, andWI-38, monkey cell lines such as COS-7 and VERO, and hamster cell linessuch as BHK-21 and CHO, all of which are publicly available from theAmerican Type Culture Collection (ATCC), Rockville, Md. 20852 USA.

Expression vectors, unlike cloning vectors, should contain a promoterthat is recognized by the host organism and is operably linked to theHoxCG nucleic acid. Promoters are untranslated sequences that arelocated upstream from the start codon of a gene and that controltranscription of the gene (that is, the synthesis of mRNA). Promoterstypically fall into two classes, inducible and constitutive. Induciblepromoters are promoters that initiate high level transcription of theDNA under their control in response to some change in cultureconditions, for example, the presence or absence of a nutrient or achange in temperature.

A large number of promoters are known, that may be operably linked toHoxCG DNA to achieve expression of HoxCG in a host cell. This is not tosay that the promoter associated with naturally occurring HoxCG DNA isnot usable. However, heterologous promoters generally will result ingreater transcription and higher yields of expressed HoxCG.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoters, Goeddel et al., Nature 281:544-548(1979), tryptophan (trp) promoter, Goeddel et al., Nuc. Acids Res.8:4057-4074 (1980), and hybrid promoters such as the tac promoter,deBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983). However,other known bacterial promoters are suitable. Their nucleotide sequenceshave been published, Siebenlist et al., Cell 20:269-281 (1980), therebyenabling a skilled worker operably to ligate them to DNA encoding HoxCGusing linkers or adaptors to supply any required restriction sites. SeeWu et al., Meth. Enz. 152:343-349 (1987).

Suitable promoters for use with yeast hosts include the promoters for3-phosphoglycerate kinase, Hitzeman et al., J. Biol. Chem.255:12073-12080 (1980); Kingsman et al., Meth. Enz. 185:329-341 (1990),or other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Dodson et al., Nuc. Acids res. 10:2625-2637 (1982); Emr,Meth. Enz. 185:231-279 (1990).

Expression vectors useful in mammalian cells typically include apromoter derived from a virus. For example, promoters derived frompolyoma virus, adenovirus, cytomegalovirus (CMV), and simian virus 40(SV40) are commonly used. Further, it is also possible, and oftendesirable, to utilize promoter or other control sequences associatedwith a naturally occurring DNA that encodes HoxCG, provided that suchcontrol sequences are functional in the particular host cell used forrecombinant DNA expression. In particular, in the present invention itmay be desirable to utilize the zBMP2 promoter or SMAD promoter orgoosecoid promoter such that a spatio-temporal expression of the HoxCGoccurs.

Other control sequences that are desirable in an expression vector inaddition to a promoter are a ribosome-binding site, and in the case ofan expression vector used with eukaryotic host cells, an enhancer.Enhancers are cis-acting elements of DNA, usually about from 10-300 bp,that act on a promoter to increase the level of transcription. Manyenhancer sequences are now known from mammalian genes (for example, thegenes for globin, elastase, albumin, α-fetoprotein and insulin).Typically, however, the enhancer used will be one from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See Kriegler, Meth. Enz. 185:512-527(1990).

Expression vectors may also contain sequences necessary for thetermination of transcription and for stabilizing the messenger RNA(mRNA). Balbas et al., Meth. Enz. 185:14-37 (1990); Levinson, Meth. Enz.185:485-511 (1990). In the case of expression vectors used witheukaryotic host cells, such transcription termination sequences may beobtained from the untranslated regions of eukaryotic or viral DNA's orcDNAs. These regions contain polyadenylation sites as well astranscription termination sites. Birnsteil et al., Cell 41:349-359(1985).

In general, control sequences are DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostcell. “Expression” refers to transcription and/or translation. “Operablylinked” refers to the covalent joining of two or more DNA sequences, bymeans of enzymatic ligation or otherwise, in a configuration relative toone another such that the normal function of the sequences can beperformed. For example, DNA for a pre-sequence or secretory leader isoperably linked to DNA for a polypeptide if it is expressed as apreprotein that participates in the secretion of the polypeptide; apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Generally, “operably linked” means that the DNAsequences being linked are contiguous and, in the case of a secretoryleader, contiguous and in reading frame. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,then synthetic oligonucleotide adaptors or linkers are used, inconjunction with standard recombinant DNA methods.

Expression and cloning vectors also will contain a sequence that enablesthe vector to replicate in one or more selected host cells. Generally,in cloning vectors this sequence is one that enables the vector toreplicate independently of the host chromosome(s), and includes originsof replication or autonomously replicating sequences. Such sequences arewell known for a variety of bacteria, yeast, and viruses. The origin ofreplication from the plasmid pBR322 is suitable for most gram-negativebacteria, the 2 μ plasmid origin is suitable for yeast, and variousviral origins (for example, from SV40, polyoma, or adenovirus) areuseful for cloning vectors in mammalian cells. Most expression vectorsare “shuttle” vectors, i.e. they are capable of replication in at leastone class of organisms but can be transfected into another organism forexpression. For example, a vector may be cloned in E. coli and then thesame vector is transfected into yeast or mammalian cells for expressioneven though it is not capable of replicating independently of the hostcell chromosome.

The expression vector may also include an amplifiable gene, such as thatcomprising the coding sequence for dihydrofolate reductase (DHFR). Cellscontaining an expression vector that includes a DHFR gene may becultured in the presence of methotrexate, a competitive antagonist ofDHFR. This leads to the synthesis of multiple copies of the DHFR geneand, concomitantly, multiple copies of other DNA sequences comprisingthe expression vector, Ringold et al., J. Mol. Apl. Genet. 1:165-175(1981), such as a DNA sequence encoding HoxCG. In that manner, the levelof HoxCG produced by the cells may be increased.

DHFR protein encoded by the expression vector also may be used as aselectable marker of successful transfection. For example, if the hostcell prior to transformation is lacking in DHFR activity, successfultransformation by an expression vector comprising DNA sequences encodingHoxCG and DHFR protein can be determined by cell growth in mediumcontaining methotrexate. Also, mammalian cells transformed by anexpression vector comprising DNA sequences encoding HoxCG, DHFR protein,and aminoglycoside 3′ phosphotransferase (APH) can be determined by cellgrowth in medium containing an aminoglycoside antibiotic such askanamycin or neomycin. Because eukaryotic cells do not normally expressan endogenous APH activity, genes encoding APH protein, commonlyreferred to as neo^(r) genes, may be used as dominant selectable markersin a wide range of eukaryotic host cells, by which cells transfected bythe vector can easily be identified or selected. Jiminez et al., Nature,287:869-871 (1980); Colbere-Garapin et al., J. Mol. Biol. 150:1-14(1981); Okayama & Berg, Mol. Cell. Biol., 3:280-289 (1983).

Many other selectable markers are known that may be used for identifyingand isolating recombinant host cells that express HoxCG. For example, asuitable selection marker for use in yeast is the trp1 gene present inthe yeast plasmid YRp7. Stinchcomb et al., Nature 282:39-43 (1979);Kingsman et al., Gene 7:141-152 (1979); Tschemper et al., Gene10:157-166 (1980). The trp1 gene provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan, forexample, ATCC No. 44076 or PEP4-1 (available from the American TypeCulture Collection, Rockville, Md. 20852 USA). Jones, Genetics 85:12(1977). The presence of the trp1 lesion in the yeast host cell genomethen provides an effective environment for detecting transformation bygrowth in the absence of tryptophan. Similarly, Leu2-deficient yeaststrains (ATCC Nos. 20622 or 38626) are complemented by known plasmidsbearing the Leu2 gene.

Particularly useful in the invention are expression vectors that providefor the transient expression in mammalian cells of DNA encoding HoxCG.In general, transient expression involves the use of an expressionvector that is able to efficiently replicate in a host cell, such thatthe host cell accumulates many copies of the expression vector and, inturn, synthesizes high levels of a desired polypeptide encoded by theexpression vector. Transient expression systems, comprising a suitableexpression vector and a host cell, allow for the convenient positiveidentification of polypeptides encoded by cloned DNA's, as well as forthe rapid screening of such polypeptides for desired biological orphysiological properties. Yang et al., Cell 47:3-10 (1986); Wong et al.,Science 228:810-815 (1985); Lee et al., Proc. Nat Acad. Sci. USA82:4360-4364 (1985). Thus, transient expression systems are particularlyuseful in the invention for expressing DNA's encoding amino acidsequence variants of HoxCG, to identify those variants which arefunctionally active.

Since it is often difficult to predict in advance the characteristics ofan amino acid sequence variant of HoxCG, it will be appreciated thatsome screening of such variants will be needed to identify those thatare functionally active. Such screening may be performed in vitro, usingroutine assays for receptor binding, or assays for cell proliferation,cell differentiation or cell viability, or using immunoassays withmonoclonal antibodies that selectively bind to HoxCG that effect thefunctionally active HoxCG, such as a monoclonal antibody thatselectively binds to the active site or receptor binding site of HoxCG.

As used herein, the terms “transformation” and “transfection” refer tothe process of introducing a desired nucleic acid, such a plasmid or anexpression vector, into a host cell. Various methods of transformationand transfection are available, depending on the nature of the hostcell. In the case of E. coli cells, the most common methods involvetreating the cells with aqueous solutions of calcium chloride and othersalts. In the case of mammalian cells, the most common methods aretransfection mediated by either calcium phosphate or DEAE-dextran, orelectroporation. Sambrook et al., eds., Molecular Cloning, pp. 1.74-1.84and 16.30-16.55 (Cold Spring Harbor Laboratory Press, 1989). Followingtransformation or transfection, the desired nucleic acid may integrateinto the host cell genome, or may exist as an extrachromosomal element.

Host cells that are transformed or transfected with the above-describedplasmids and expression vectors are cultured in conventional nutrientmedia modified as is appropriate for inducing promoters or selecting fordrug resistance or some other selectable marker or phenotype. Theculture conditions, such as temperature, pH, and the like, suitably arethose previously used for culturing the host cell used for cloning orexpression, as the case may be, and will be apparent to those skilled inthe art.

Suitable host cells for cloning or expressing the vectors herein areprokaryotes, yeasts, and higher eukaryotes, including insect, oysters,lower vertebrate, and mammalian host cells. Suitable prokaryotes includeeubacteria, such as Gram-negative or Gram-positive organisms, forexample, E. coli, Bacillus species such as B. subtilis, Pseudomonasspecies such as P. aeruginosa, Salmonella typhimurium, or Serratiamarcescans.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable hosts for zBMP2, HoxCG and blockermolecule-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe, Beach and Nurse, Nature 290:140-142 (1981), Pichia pastoris,Cregg et al., Bio/Technology 5:479-485 (1987); Sreekrishna, et al.,Biochemistry 28:4117-4125 (1989), Neurospora crassa, Case, et al., Proc.Natl. Acad. Sci. USA 76:5259-5263 (1979), and Aspergillus hosts such asA. nidulans, Ballance et al., Biochem. Biophys. Res. Commun. 112:284-289(1983); Tilburn et al., Gene 26:205-221 (1983); Yelton et al., Proc.Natl. Acad. Sci. USA 81:1470-1474 (1984), and A. niger, Kelly et al.,EMBO J. 4:475-479 (1985).

Suitable host cells for the expression of HoxCG also are derived frommulticellular organisms. Such host cells are capable of complexprocessing and glycosylation activities. In principle, any highereukaryotic cell culture is useable, whether from vertebrate orinvertebrate culture. It will be appreciated, however, that because ofthe species-, tissue-, and cell-specificity of glycosylation, Rademacheret al., Ann. Rev. Biochem. 57:785-838 (1988), the extent or pattern ofglycosylation of HoxCG in a foreign host cell typically will differ fromthat of HoxCG obtained from a cell in which it is naturally expressed.

Examples of invertebrate cells include insect and plant cells. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori host cells have been identified. Luckow etal., Bio/Technology 6:47-55 (1988); Miller et al., in GeneticEngineering, vol. 8, pp.277-279 (Plenum Publishing, 0.1986); Maeda etal., Nature 315:592-594 (1985).

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens. During incubation of the plant cells with A.tumefaciens, the DNA is transferred into cells, such that they becometransfected, and will, under appropriate conditions, express theintroduced DNA. In addition, regulatory and signal sequences compatiblewith plant cells are available, such as the nopaline synthase promoterand polyadenylation signal sequences, and the ribulose biphosphatecarboxylase promoter. Depicker et al., J. Mol. Appl. Gen. 1:561-573(1982). Herrera-Estrella et al., Nature 310:115-120 (1984). In addition,DNA segments isolated from the upstream region of the T-DNA 780 gene arecapable of activating or increasing transcription levels ofplant-expressible genes in recombinant DNA-containing plant tissue.European Pat. Pub . . . . No. EP 321,196 (published Jun. 21, 1989).

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure in recent years. Kruse & Patterson, eds., Tissue Culture(Academic Press, 1973). Examples of useful mammalian host cells are themonkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line 293 (or 293 cells subcloned for growth insuspension culture), Graham et al., J. Gen Virol. 36:59-72 (1977); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells(including DHFR-deficient CHO cells, Urlaub et al., Proc. Natl. Acad.Sci. USA 77:421.6-4220 (1980); mouse sertoli cells (TM4, Mather, Biol.Reprod. 23:243-251 (1980); monkey kidney cells (CV1, ATCC CCL 70);African green monkey kidney cells (VERO-76, ATCC CRL-1587); humancervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and ahuman hepatoma line (Hep G2).

Construction of suitable vectors containing the nucleotide sequenceencoding HoxCG and appropriate control sequences employs standardrecombinant DNA methods. DNA is cleaved into fragments, tailored, andligated together in the form desired to generate the vectors required.

For analysis to confirm correct sequences in the vectors constructed,the vectors are analyzed by restriction digestion (to confirm thepresence in the vector of predicted restriction endonuclease) and/or bysequencing by the dideoxy chain termination method of Sanger et al.,Proc. Nat. Acad. Sci. USA 72:3918-3921 (1979).

The mammalian host cells used to produce the HoxCG of this invention maybe cultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham, et al., Meth. Enz. 58:44-93 (1979); Barnes et al.,Anal. Biochem. 102:255-270 (1980); Bottenstein et al., Meth. Enz.58:94-109 (1979); U.S. Pat. Nos. 4,560,655; 4,657,866; 4,767,704; or4,927,762; or in PCT Pat. Pub. Nos. WO 90/03430 (published Apr. 5,1990), may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleosides (such as adenosine and thymidine),antibiotics, trace elements (defined as inorganic compounds usuallypresent at final concentrations in the micromolar range), and glucose oran equivalent energy source. Any other necessary supplements may also beincluded at appropriate concentrations that would be known to thoseskilled in the art. The culture conditions, such as temperature, pH, andthe like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.

The host cells referred to in this disclosure encompass cells in culturein vitro as well as cells that are within a host animal, for example, asa result of transplantation or implantation.

It is further contemplated that the HoxCG of this invention may beproduced by homologous recombination, for example, as described in PCTPat. Pub. No. WO 91/06667 (published May 16, 1991). Briefly, this methodinvolves transforming cells containing an endogenous gene encoding HoxCGwith a homologous DNA, which homologous DNA comprises (1) an amplifiablegene, such as DHFR, and (2) at least one flanking sequence, having alength of at least about 150 base pairs, which is homologous with anucleotide sequence in the cell genome that is within or in proximity tothe gene encoding HoxCG. The transformation is carried out underconditions such that the homologous DNA integrates into the cell genomeby recombination. Cells having integrated the homologous DNA then aresubjected to conditions which select for amplification of theamplifiable gene, whereby the HoxCG gene amplified concomitantly. Theresulting cells then are screened for production of desired amounts ofHoxCG. Flanking sequences that are in proximity to a gene encoding HoxCGare readily identified, for example, by the method of genomic walking,using as a starting point the HoxCG nucleotide sequence set forth in SEQID NO.:23 and SEQ ID NO.:24. See Spoerel et al., Meth. Enz. 152:598-603(1987).

Gene amplification and/or gene expression may be measured in a sampledirectly, for example, by conventional Southern blotting to quantitateDNA, or Northern blotting to quantitate mRNA, using an appropriatelylabeled oligonucleotide hybridization probe, based on the sequencesprovided herein. Various labels may be employed, most commonlyradioisotopes, particularly ³²P. However, other techniques may also beemployed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radioisotopes, fluorophores, chromophores, or the like.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. The antibodies in turn may be labeledand the assay may be carried out where the duplex is bound to a surface,so that upon the formation of duplex on the surface, the presence ofantibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of the gene product, HoxCG. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled, where the labels are usually visually detectable,such as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hsu et al., Am. J. Clin. Path.,75:734-738 (1980). Antibodies useful for immunohistochemical stainingand/or assay of sample fluids may be either monoclonal or polyclonal.Conveniently, the antibodies may be prepared against a synthetic peptidebased on the DNA sequences provided herein.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises”, means “including but not limited to” and is not intended toexclude other additives, components, integers or steps.

The invention will now be further described by way of reference only tothe following non-limiting examples. It should be understood, however,that the examples following are illustrative only, and should not betaken in any way as a restriction on the generality of the inventiondescribed above. Amino acid sequences referred to herein are given instandard single letter code.

EXAMPLE 1 Isolation of Stage-Specific Promoters for a Sterile FeralConstruct

In order to identify a good candidate promoter and/or gene for theproposed construct, the applicant examined a number of animals, bothvertebrate and invertebrate. The applicant finally decided on thewell-studied model for fish, the zebrafish (Brachydanio rerio). Thisfish model was chosen as it is reasonably well characterized, and thefish are small and relatively easily breed and reared. Moreover, thezebrafish has a high degree of nucleotide and amino acid sequencehomology to most other fish species studied, and as will be shown later,a reasonably high degree of sequence homology with other non-fishspecies. This degree of similarity can permit the identification ofgenes in other species by comparison with those of zebrafish.Accordingly, it was considered, that this model was most appropriate forlocating and testing a promoter which may function across all species.At least it was a useful model for testing the broad “sterile feralconstruct” concept.

The applicant examined mutant screens in zebrafish for a target genethat was essential for a short period in larval development, but whichhad no adult functions. The applicant focused on 6 mutations that causedorso-ventral patterning defects (Mullins et al 1996), and in particularon the swirl mutant, which exhibits severe dorsalization and thecomplete lack of ventral structures such as blood and pronephros. Swirlencodes the zebrafish homologue of BMP2 and was named zBMP2 (Kishimotoet al., 1997). In zebrafish the dorsalised swirl mutant phenotype isrescued by injection of zBMP2 mRNA at the single cell stage (Kishimotoet al., 1997), which indicates that the gene is essential only duringearly larval development and plays no maternal role. BMPs (BoneMorphogenetic Proteins) are a subfamily of the larger transforminggrowth factor beta (TGF-β) superfamily of signalling molecules that playa central role in establishing the early animal body plan and inorganogenesis (Hogan, 1996).

The cDNA for the zBMP2 gene was obtained from M. Hammerschmidt (MaxPlank Institute, Frieburg) as a 1,732 bp fragment subcloned into aplasmid designated pzBMP2b. This plasmid was transformed into XL-1 bluestrain of E. coli according to the instructions of the supplier(Stratagene). A resulting positive clone carrying the plasmid was grownaccording to standard protocols, and the cDNA from the bacterial culturewas isolated by standard procedures. After digestion with EcoRI, a 422bp fragment spanning the 5′ untranslated region was isolated andlabelled with ³²P. This was then used as a probe for a zebrafish genomiclibrary.

The zebrafish genomic BAC library was purchased in the form of arrayedfilter sets, from Genome Systems Inc (GSI), and screened using thelabelled probe by standard hybridization techniques as describedpreviously. Five positive clones (BMP-BAC5, BMP-BAC10, BMP-BAC15,BMP-BAC17, and BMP-BAC21) were then purchased from Genome Systems Inc(GSI). Preliminary sequencing of all five positive BAC clones usingprimers specific of the 5′-untranslated region of the cDNA revealed thatthe clones were identical to each other and to the region of the BMP2cDNA. Two of the BAC clones (BMP-BAC5 and BMP-BAC10) were subcloned asHindIII fragments into pGEM-7ZF(+) by standard procedures. We obtained6,915 bp of sequence from these clones which represented from −3879 to+3035 bp relative to the translation start site. The coding sequenceobtained was identical to the zebrafish zBMP2 cDNA sequence previouslydescribed by Nikido et al. (1997) and Lee et al. (1998). This suggestedthat BAC 5 and 10, and perhaps the remaining three BAC clones, containedauthentic zebrafish BMP2 genomic DNA. However, based on the genomicsequences we obtained, the previously designated start site, at 376 bpin the cDNA (Lee et al., 1998), lies in the second exon and the firstexon is untranslated.

Further definition and isolation of the zBMP2 promoter was accomplishedby sequencing these HindIII subclones to isolate candidate fragmentswhich resided 5′ of the sequence homologous to the cDNA coding for zBMP2gene. One of these subclones had a 5,901 bp insert that was positive forzBMP2 gene. FIG. 1 shows the resultant plasmid pBAC5/H11. The insert wasalso found to include a 1,414 bp region that was 5′ of the presumptivestart codon of zBMP2, and which was considered to be a possible locationof the zBMP2 promoter. A 1,414 bp fragment was excised from pBAC5/H11with SmaI/EcoRI and subcloned into the multiple cloning site ofpBluescript-II-SK. This fragment contained the putative zBMP2 promoterfrom about 60 bp 5′ of the first splice site. A SacI-KpnI fragment wasthen excised from this plasmid and directionally cloned into pGEM-EGFPcontaining the modified GFP reporter gene (GM2, see Cormack et al.,1996) resulting in the construct pzBMP2(1.4)-EGFP as shown in FIG. 2.

We considered that the control of expression of zBMP2 gene likelyresided in this SacI-KpnI fragment, and would be useful in controllingthe “Sterile-Feral” construct. However, we are sure that any promoterwith an appropriate spatial-temporal pattern could be used in the final“Sterile-Feral” construct. The construct pzBMP2(1.4)-EGFP was insertedinto zebrafish embryos to test whether it followed a similarspatial-temporal expression pattern as reported for the zBMP2 promoter.

This construct and all subsequent constructs were prepared using thefollowing procedures and introduced into the developing embryos bymicroinjection.

All the DNA preparations were appropriately linearized and gel purified(Qiaquick Gel Extraction Kit) before injection. Needles were made fromborosilicate glass capillaries with filaments (GC100TF-15, ClarkElectromedical instruments) using a P-80PC micropipette puller (SutterInstrument Co.). The needle was back-filled with purified DNA diluted to100 ng/μl in 1× injection buffer (10×; 50 mM Tris; 5 mM EDTA; 1M KCl,pH7.2) using a hand pulled pipette. Injections were carried out on adissection microscope fitted with two, 3-dimensional Narshige MN-151micromanipulators. Embryos were held in place during injection by ahydraulically (mineral oil) driven holding pipette. Injection of DNAsolution was facilitated pneumatically using a 3-way foot operatedplunge valve (Festo Engineering), connected between the injection needleholder and nitrogen tank. Injection was performed on one-cell stageembryos, unless specifically indicated otherwise. Injected embryos wereincubated and reared as described above.

Post-injection, early-stage embryos were examined under UV illuminationin a Zeiss microscope equipped with standard fluorescent isothiocynate(FITC) filter set, while later-stage embryos were anaesthetized inembryo medium containing 0.125%, 2-phenoxyethanol (Sigma P-1126), beforeexamination. Photomicrographs of embryos expressing EGFP were obtainedfor analysis.

Table 1 summarises the injection trials. The percentage of embryosexpressing EGFP at 10 h post injection (pi), varied from batch to batch,ranging from 0% to 42.7%.

Expression was detectable as early as dorsal shield stage (6 h pi) inmost of the expressing embryos. At 9.5 h pi, the majority of theexpressing embryos had expression that was limited to anterior ventralregions (FIG. 3 a); however, 3 embryos expressed EGFP all along theventral margin (FIG. 4 a). The patchiness is typical of the mosaicexpression expected in founder transgenic animals. Nonetheless,expression domains extended from polster region (FIG. 3 a;PO) anteriorlyto the region of future tail bud, posteriorly (FIG. 3 a;TB). TABLE 1Results of EGFP expression in embryos injected with pzBMP2(1.4)-EGFP atabout 9.5-10 h Post Injection Number with No. with Total No. Anterio-entire Number with ventral ventral Batch Observed Expression expressiondomain 1 28 0 0 0 2 21 3 1 2 3 20 2 0 2 4 28 12 2 10

At about 24 h pi, expression was predominantly in the ventral domains(FIG. 5 a), mimicking the native zBMP2 expression—in the region of thedeveloping eye, otic vesicle, and pectoral fin bud. Abolition of tailbud expression at 24 h pi suggests that the cloned promoter may lackregulatory elements responsible for maintenance of BMP2 expression atthis stage. No EGFP expression was detected by 48 h pi, suggesting thatthe zBMP2 gene is not required this late in development.

The zBMP2 promoter sequence is shown in SEQ ID NO:1.

EXAMPLE 2 Isolation of Second Promoter for Sterile Feral Construct

As the applicant was concerned about the potential shortcomings/delaysof the BMP2 promoter in combination with a tet-responsive (tetOff)element to effectively block its own native transcripts, an earlyacting, but temporally restricted promoter sharing spatial domains withthat of BMP2 was considered preferable. One such candidate was thezebrafish SMAD5. Similar to BMP2, mutation in the zebrafish SMAD5results in a dorsalized mutation designated somitabun (sbn) and thedorsalised mutant phenotype has been shown to be rescued by injection ofSMAD5 mRNA at the single cell stage (Hild et al., 1999). This indicatedthat the gene is essential only during early larval development. It hasalso been implied that the SMAD5 acts as a transducer of BMP2 signallingwith potential upstream and downstream functions. The functionalassociation between the BMP2 and SMAD5 suggested that the two genesshare the same spatial expression domains. Further the maternalexpression of SMAD5 and also the relative early onset of zygotic SMAD5expression ensure that the cells are competent to process BMP2signalling (Hild et al., 1999; Dick et al., 1999). Therefore, weconsidered that by employing a SMAD5 promoter to drive the expression ofa BMP2 blocker would alleviate some of the potential temporal delaysassociated with employing the BMP2 promoter.

The cDNA for the SMAD5 gene was amplified from zebrafish shield stagecDNA using following primers SMADu1: 5′-TGCAGGTGGACTTTGGATCCG-3′ SEQ.ID. NO.: 4 SMADL1: 5′-GCCTAAAGGCAACAGATGCTA-3′ SEQ. ID. NO.: 5

The primers were designed based on the published zebrafish SMAD5 cDNAsequences (Hild et al., 1999). The amplified 2285 bp product was clonedinto pGem-T-Easy vector as per the cloning instructions of themanufacturer (Promega, Madison USA) and confirmed by sequencing. Aresulting positive clone carrying the plasmid was grown according tostandard protocols, and the cDNA from the bacterial culture was isolatedby standard procedures. A 366 bp fragment spanning the 5′ untranslatedregion was isolated and labelled with ³²P. This was then used as a probefor a zebrafish genomic library.

Four positive clones (SMAD-BAC1, SMAD-BAC8, SMAD-BAC13, and SMAD-BAC 17)were then purchased from GSI. Preliminary sequencing of all fourpositive BAC clones using primers specific of the 5′-untranslated regionof the cDNA revealed that the clones were identical to each other and tothe region of the BMP2 cDNA. One of the BAC clones (SMAD-BAC51) wassubcloned as HindIII fragments into pGEM-7ZF(+) by standard procedures.We obtained a positive subclone of about 8 KB (psBAC1/H12), thatcontained 1,005 bp of putative promoter sequence 5′ of the start codon.The coding sequence obtained was identical to the zebrafish SMAD5 cDNAsequence previously described by Hild et al. (1999).

A 1,005 bp putative promoter fragment was then amplified from psBAC1/H12with the following primers M13 forward: 5′-GTAAAACGACGGCCAGT SEQ ID NO:6 SMAD L2: 5′-TAGTGCTGGGCTGCACCAG SEQ ID NO: 7

The amplified fragment was ligated into pGEM-Teasy vector and theorientation and sequence confirmed (pSMAD5′). The promoter was againexcised as SmaI/EcoRI fragment, blunt ended and ligated into the SmaIlinearized pGEM-EGFP. A positive clone, pSMAD5-EGFP (FIG. 6) in thecorrect orientation was selected and tested in vivo in zebrafishembryos.

Injection trials of pSMAD5-EGFP into the zebrafish embryo resulted inexpression of the EGFP as early as 4 hp. The expression pattern wasubiquitous initially as late as shield stage (FIG. 7), thenpredominantly restricting to ventral tissues at about 24 hpi (FIG. 8).The experimental evidence suggested that the zygotic expression of SMAD5was activated marginally ahead of zBMP2. Although preliminary, ourpromoter analysis experiments suggested that the SMAD5 promoter wasindeed activated slightly ahead of bmp2 promoter (data not shown). NoEGFP expression was detected by 48 hpi, suggesting that the SMAD5 genewas not required this late in development.

The zebrafish SMAD5 promoter sequence is shown in SEQ ID NO; 8.

EXAMPLE 3 Zebrafish Model

Breeding and rearing protocols for zebrafish generally followWesterfield (1995). Stock was obtained from a local pet store; however,it would be appreciated by those skilled in the art that zebrafish couldequally be obtained from laboratories around the world (e.g., Instituteof Neuroscience, eugene, Oreg., USA) and maintained at 27-28° C. in anin-house re-circulatory flow-through system. Embryos were obtained bynatural matings, transferred into Embryo Medium (Westerfield, 1995), andincubated in a bench top incubator at 26-27° C. until 3-4 days old. Theywere then transferred into nursery tanks maintained at 27-28° C., andreared on finely ground commercial fish flakes (Tetramin), and liveArtemia. After approximately 3 months, the fish were transferred intostandard fish tanks alongside the adult fish. The adult fish were feddaily with flakes and occasionally supplemented with either freshlyhatched or frozen Artemia.

EXAMPLE 4 Blocking Expression of zBMP2

The applicant tested three options for blocking expression of thecandidate genes: mis/over-expression of sense (see below), antisense(Izant and Weintraub 1984) and double stranded RNA (dsRNA). (Guo andKemphues, 1995). The latter appears to be more potent than antisense atinducing interference in C. elegans (Fire et al., 1998) and has beenemployed to silence native and reporter genes in plants (Waterhouse etal., 1998). To develop and optimise the blocking component of the“sterile feral” construct, the applicant assayed sense, antisense, anddsRNA of zBMP2 by injection in zebrafish embryos. Results indicated thatboth antisense and dsRNA block gene expression, whereas sense strandinjection resulted in over-expression.

Capped full-length sense and antisense zBMP2 RNA transcripts weregenerated by linearizing the plasmid pzBMP2b, whereas the truncatedversions of just the 5′- or the 3′-regions were generated byappropriately linearised pzBMP2-ApaI or pzBMP2-BstXI, respectively. Allin vitro transcriptions were carried out using T3/T7 mMESSAGE mMACHINE™(Ambion), as appropriate. dsRNA was prepared by annealing sense andantisense RNA in RNAase free injection buffer at 37° C. for 5 minutesfor the truncated and 10 minutes for the full-length transcripts.Annealing of respective sense and antisense strands as dsRNA wasconfirmed by running a sample on a non-denaturing agarose gel. About 3-5picolitres of RNA solutions, ranging between 100-250 ng/μl, wereinjected into 1-2 cell stage embryos as described above in Example 1. Inthe case of 2-cell stage injections, both the cells were injected.

In embryos injected with full-length antisense or dsRNA of zBMP2, theproportion of normal embryos was significantly reduced and some weaklydorsalised embryos resembling zebrafish swirl mutant were seen (FIG. 9a&b). Sense injections resulted in mild ventralization of the embryos,which in some cases resembled the zebrafish chordino mutant phenotype(FIG. 10). Chordino is the dorsally expressing zebrafish homologue ofchordin, known to interact antagonistically with BMPs (in this caseswirl) in a dose dependent manner (Kishimoto et al., 1997).

To obtain molecular data to support hypothesised interference of thedsRNA on expression of zBMP2, the applicant injected truncated forms ofzBMP2 ds RNA, so as to use the uninjected portion as probe to detect andquantify the native transcript levels in the injected embryos. Thepercentage of deformed embryos in groups injected with 3′-zBMP2 and5′-zBMP2 dsRNA was 43.4% and 40.2%, as compared to 9.2% and 2.4% in thecorresponding controls (Table 2). TABLE 2 Results of Truncated zBMP2dsRNA Injection Into One-Cell Stage Embryos Transcript Number NumberNumber Injected Conc. ng/μl injected Survivors* deformed* 3′-zBMP2 150123 83 36 (67.5) (43.4) Control 0 66 54  5 (81.8)  (9.2) 5′-zBMP 250 8867 27 (76.1) (40.2) Control 0 53 42  1 (79.2)  (2.4)*Results in parenthesis indicate percentages

EXAMPLE 5 Combined Promoter and Blocker DNA Construct

On confirming the ability of in vitro transcribed BMP2 antisense anddouble stranded transcripts to disrupt larval development, DNAconstructs capable of expressing the antisense and double strandedtranscripts in vivo were developed and tested.

A 711 bp ApaI fragment of the zBMP2 cDNA was excised from the plasmidpzBMP2b and inserted into the ApaI linearized pzBMP2(1.4)-EGFP resultingin the pzBMP2As-EGFP (FIG. 11). Antisense orientation of zBMP2 fragmentin pzBMP2AS-EGFP was confirmed both by restriction analysis andsequencing. The pzBMP2As-EGFP was a fusion construct capable ofco-expressing BMP2 antisense and EGFP. Co-expression of EGFP with theBMP2 antisense provided an easily detectable marker to distinguish themutant embryos emanating from antisense interference and thosepotentially resulting from spontaneous or background mutations.pzBMP2As-EGFP was linearized with NotI for injection into the embryos.

For the double stranded knockout, four segments of the zBMP2 gene werearranged to express double stranded mRNA in vivo (FIG. 12). Thefirst-section comprised the 1,414 bp “HindIII-EcoRI” promoter regionretained in the pGEM 7zf(+) vector backbone, obtained by excising theEcoRI-SacI coding region of the zBMP2 from pBAC5/H11 subclone. Thesecond segment was a 510 bp fragment of the zBMP2 cDNA from sequence301-810 in the published cDNA sequence (Lee et al., 1998). This fragmentwas amplified using the following primers: zfEx1-3.EcoF Forward Primer5′-ACCCCGAATTCATGAGGAACTTAGGA-3′ SEQ ID NO: 9 zfEx1-3.SalR ReversePrimer 5′-ATCAGCTCGTCGACAGGAATGGAGGTAAG-3′ SEQ ID NO: 10

The amplified product generated had an EcoRI site on the 5′-end and aSalI site on the 3′-end for ease of cloning. The third section was a 286bp fragment of cDNA (bases 307-592) which was amplified using thefollowing primers: Bex1i.PstF 2 Forward Primer5′-ACACCTGCAGATGAGGAACTTAGGAGACGAC-3′ SEQ ID NO: 11 Bex1i.SalR ReversePrimer 5′-TACTGAGGGTCGACTGCCGATTTGCT-3′ SEQ ID NO: 12

These primers generated a PstI site on the 5′ end and SalI site on the3′ end for cloning. When ligated to the second fragment, the thirdsegment formed an inverted repeat of the 5′ end of the cDNA (bases 307through 592). The final segment was a PstI-SacI fragment containing apoly A tail section, excised from the pGT2-ns-GM2f construct that waskindly donated by Dr. Shou Lin, Institute of Molecular Medicine andGenetics, Medical College of Georgia. The DNA sequence for the doublestranded BMP2 construct is given as SEQ ID NO:13.

Results of the BMP2 antisense-EGFP fusion construct injection arepresented in the Table 3. TABLE 3 Results of NotI linearizedpzBMP2-As-EGFP Injection into the One-Cell Zebrafish Embryos NumberConc. Number Number Number with EGFP Batch μg/ml injected Survivors*deformed* expression 1 100 48 36 1 0 (75)    (2.7) 0 40 29 0 0 (72.5) 2100 36 16 6 5 (44.4) (37.5) (31.3) 0 16  9 0 0 (56.2) 3 100 20 12 4 3(60)   (33.3) (75)   0 23 15 0 0 (65.2)*Figures in parenthesis indicate percentages.

The number of deformed individuals in the injected groups ranged from 0%to 37.5%. The majority of the deformed individuals (83.3% and 75% inbatches 1 and 2, respectively) expressed EGFP, indicating that theantisense was effective in disrupting the larval development. None ofthe individuals in the control group and non-deformed individuals in theinjected group had EGFP expression.

Results of the zBMP2-double stranded construct are given in Table 4.TABLE 4 Results of pzBMP2-ds Injection into 1-4 Cell Stage ZebrafishEmbryos Treatment Number Number of Number Batch Conc. (μg/ml Treatedmortality Deformed Injected 0 37  4 (10.8) 0 Control Uninjected — 123 24(20.5) 1 (0.8)* control dsRNA injected 100 143 20 (14.3) 21 (14.7) Uninjected — 51 11 (17.1) 0 control dsRNA injected 100 47  7 (16.5) 22(45.7) Figures in parenthesis indicate percentages.*denotes a deformed control fish that had deformities thatdid not resemble the swirl mutants.

Of 211 control embryos (mock-injected with buffer only or permitted todevelop normally), only one embryo was deformed. The deformity did notresemble the swirl mutant. In the two dsDNA treatment groups, 14.7% and45.7% of the embryos expressed the swirl mutation.

EXAMPLE 6 The Repressible Element

The proof-of-concept used a commercially available repressible elementas the externally keyed genetic switch or Tet-responsive P_(hCMV*-1)promoter. P_(hCMV*-1) contains the Tet-responsive element (TRE) whichconsists of seven copies of the 42 bp tet operator sequence (tetO). Thiselement is just upstream of the minimal CMV promoter (P_(minCMV)), whichlacks the enhancer that is part of the complete CMV promoter. Therefore,P_(hCMV*-1) is silent in the absence of binding of transactivatorprotein (tTA) to the tetO. The tetracycline-sensitive element isdescribed by Gossen and Bujard (1992; tet-off), Gossen et al. (1995;Tet-on), and Kistner et al. (1996). In the tetracycline-regulated system(Tet-Off system) developed by Hermann Bujard, addition of tetracycline(Tc) or doxycycline Dox; a Tc derivative) prevents the binding of a tTA,to the Tet-responsive element. This then blocks gene expression from theTRE until the drug is removed. A complementary system has also beendeveloped (Tet-On system). In the Tet-On system, addition of doxycyclineallows the binding of a reverse transactivater, rtTA, to the tetOpromoter, leading to gene expression from the TRE. Gene expressioncontinues from the TRE until removal of the drug. A tetracyclineresponsive element has the advantage of ease of administering.Tetracycline is a routinely used antibiotic in fish and shellfishculture (see Stoffregan et al., 1996), readily traverses cutaneousmembranes while retaining its biological activity, and can beadministered by whole organism immersion. Use of the Tet-On/Offcontrollable expression systems is covered by U.S. Pat. No. 5,464,758,assigned to BASF Aktiengesellschaft.

The applicant first tested the functionality of the Tet-off system inzebrafish cell cultures. The cell culture was established using ZF4cells as previously described (Driever and Rangini, 1993). Cells weretransfected with the DNAs using Effectene liposomes (Qiagen) accordingto the manufacturer's instructions. Cells were initially transfectedwith pTet-Off and placed under neomycin selection for 1 month.Neomycin-resistant cells were then transfected with pTRE-EGFP, and theselection plasmid pTK-Hyg, and placed under hygromycin selection for twoweeks. EGFP expression was determined by examining and counting cellswith obvious fluorescence and by examination of cell lysates using afluorometer. Cells were grown in medium with or without doxycycline (0.2μg/ml) for 72 h prior to assessment of gene expression, or were rinsedof doxycycline and assessed for reporter gene expression 72 h afterremoval of doxycycline.

In the absence of doxycycline, EGFP fluorescence was detected in a smallpercentage (approximately 6%) of cells (Table 5). TABLE 5 Transfection %cells expressing EGFP expression in Treatment EGFP cell lysates None 0 0 ± 15 pTet-Off 0  0 ± 12 pTRE-EGFP 0 0 ± 9 pTet-Off + pTRE- 5.9 ± 1.286 ± 11 EGFP pTet-Off + pTRE- 0.2 ± 0.1 5 ± 3 EGFP + Dox (72 h)pTet-Off + pTRE- 2.6 ± 0.9 49 ± 6  EGFP + removal of Dox (72 h)Values represent the average and standard errors for 3 separatetransfection experiments, each containing 4 replicates.

The low percentage of cells expressing the reporter gene presumablyreflects the efficiency of simultaneously transfecting the cells withtwo plasmids (pTRE-EGFP and pTK-Hyg). When doxycycline was added, EGFPgene expression dropped substantially, to approximately 3% of expressionlevels seen in cells not exposed to doxycycline. Interestingly, washingthe cells and removing as much of the doxycycline as possible couldreverse the repression of reporter gene expression. Fluorometric assaysof cell lysates performed using a BMG FluoStar showed similar results tocell counts, with repression of the EGFP fluorescence being repressed inthe presence of doxycycline. The reversal of the repression followingremoval of doxycycline appeared greater in these assays, most likelybecause the fluorometer could detect low levels of fluorescence notdetected by microscopic examination.

Next the applicant tested the tet-off system in whole zebrafish embryos.The Tet-On™ and Tet-off™ gene expression system and the Tet responsivebidirectional vectors pBI and pBI-EGFP were purchased from a commercialsource (Clontech). The pzBMP2-Tet-Off construct (FIG. 13) was engineeredby excising PminCMV promoter as SpeI and EcoRI fragment from pTet-Offand replacing it with the 1,414 bp zBMP2 promoter as XbaI/EcoRI, frompzBMP2-(1.4), by directional cloning. The pzBMP2-Tet-Off and pBIconstructs were linearised with SacI and PuvII, respectively and columnpurified using a PCR purification column (Qiagen). Eluted DNA werequantified and mixed in equimolar ratio to yield a final concentrationof about 150 ng/μl in injection buffer. Injections were carried outusing one-cell stage embryos as described in Example 1.

Of the 84 embryos co-injected, EGFP expression was detectable in 7(8.3%) individuals at about 24 h pi. A low percentage of transformedembryos is typical of co-injection experiments. The spatial pattern ofEGFP expression (along the anterio-ventral regions) is similar to thatwe previously observed when EGFP was directly under the regulation ofzBMP2 promoter.

EXAMPLE 7 Complete Zebrafish Sterile Feral Construct

A single tet responsive double stranded RNA blocker construct under theregulation of zBMP2 promoter, pBIT(Bmp2)-Bmp2ds (FIG. 14), was builtusing pBI-EGFP as the backbone. The bidirectional tet responsiveconstruct with EGFP as a marker was chosen to provide a visible marker.First, the SV40 PolyA was excised from the vector pBI-EGFP (Clontech,PT3146-5) following digestion with AatII and SalI. The resultingfragment was blunt ended with T4 DNA polymerase and religated to formpBi(−SV), an intermediate plasmid.

This was then cut with HindIII and used in a subsequent ligation with aHindIII fragment containing the BMP2 promoter, which was obtained fromBMP-tetOff plasmid (SEQ ID NO:2, NM99/09099). The resulting plasmid,called pBi.tTA was then cut with with PvuII, dephosphorylated, and addedto a ligation reaction containing a second fragment (blunt ended with T4DNA polymerase), which contained the a 510 bp fragment of the zBMP2 cDNAfrom sequence 301-810 in the published cDNA sequence (Lee et al., 1998)and was obtained by digesting dsRNA (BMP2) (SEQ ID NO:13, NM99/09100)with EcoRI and HindIII followed by gel purification. This ligationreaction produced the construct pSF1. The pBIT(Bmp2)-bmp2ds construct isshown in FIG. 14 and SEQ ID NO: 14 and here through refereed to as pSF1.

Similarly pBIT(Cmv)-BMP2ds (pSF2), a zbmp2 double stranded RNA blockerconstruct in which the tet-Off (tTA) is under the regulation of CMVpromoter, was built as follows. Commercially purchased pTet-Offconstruct was digested with HindIII, XhoI and SapI. A 2250 bpXhoI/HindIII fragment containing CMV promoter, tTA and SV40 PolyA and a2000 bp SapI/XhoI fragment containing vector backbone were gel purified.Meanwhile the pBIT(bmp)-bmp2ds was digested with HindIII/SapI and a3,459 bp fragment containing EGFP and double stranded bmp2 RNA, withβ-globin poly A was gel purified. Finally the three fragments wereligated directionally to yield the pBIT(CMV)-bmp2ds (pSF2, FIG. 15, SEQID NO:15) construct.

The applicant constructed two more candidate sterile feral constructs,with tTA driven by the zebrafish SMAD5 promoter: one used BMP2 doublestranded RNA as developmental blocker [pBIT(smad)-BMP2ds] and anotherused zBMP2 sense, to be mis-expressed, as a blocker[pBIT(smad)-BMP2sense). An intermediate construct, pSmadTet-Off, wasbuilt by excising the CMVmin1 promoter as XbaI and SpeI fragmet frompTet-Off and replacing it with a 965 bp zebrafish SMAD5 promoter.

Subsequently, pBIT(smad)-BMP2ds (pSF3, FIG. 16, SEQ ID NO:16) was madeby excising CMV promoter as a XhoI/SphI fragment from pBIT(CMV)-bmp2dsand replacing it with XhoI/SphI SMAD5 promoter fragment frompSmadTet-Off. The construct was confirmed by restriction analysis andsequencing. The construct was renamed pSF3.

The pBIT(smad)-BMP2sense(pSF4, FIG. 17; SEQ ID NO:17) was constructed asfollows. Firstly a 1,440 bp zebrafish BMP2 cDNA was excised as EcoRI andXhoI fragment from pzBMP2b, blunt ended and ligated into PvuIIlinearized pBI-EGFP. The sense orientation of the bmp2 cDNA in thebi-directional vector was confirmed by restriction analysis andsequencing. A resulting clone (pBI-bmp2-Sense) in the correctorientation was prepared for further use. The double stranded RNAblocker in the pBIT(smad)-bmp2ds (pSF3) was excised as EagI/MluIfragment and replaced with EagI/MluI fragment from pBI-bmp2-Senseconstruct. The resulting pBIT(smad)-bmp2-Sense construct (pSF4, FIG. 17and SEQ ID NO:17) was confirmed by restriction analysis and sequencing.

Table 6 summarises the pooled results of three different batches of pSF1construct injections into zebrafish embryos. TABLE 6 Results of pSF1(100 ng/μl) injections into the zebrafish embryo. No No. dead dead No.No. Glow No. Non Glow Treatment Total 5 hpi 24 hpi Live Deformed NormalDeformed Normal SF1 166 65 11 90 2 34 0 52 Injected (54.2) (2.2) (37.7)(57.7) Buffer 143 56 17 70 0  0 0  0 Control (48.9)Although about 40% of the embryos had EGFP expression, only 2.2% had theassociated deformity resembling the dorsalized swirl mutation. This isin stark contrast to 14-40% swirl like deformities the applicantobserved by injection of a double stranded RNA construct (pzBMP2-ds)that was driven directly by the BMP2 promoter. The lack of correlationbetween the deformity and EGFP expression may be attributed to severalreasons, including the delay associated with the indirect expression ofthe blocker by the BMP2 promoter mediated via the expression of tTA.

Table 7 summarises the results of pSF2 injected into the embryos ofzebrafish. TABLE 7 Results of injecting pSF2 (100 ng/μl) into theembryos of zebrafish. No No. dead dead No. No. Glow No. Non GlowTreatment Total 5 hpi 24 hpi Live Deformed Normal Deformed Normal SF2Dox 175 44 30 101  3 8 6 84  (57.7)  (2.9)  (7.9) (5.9) (83.1) No Dox183 28 53 102  11  49  2 40  (55.7) (10.7) (48.0) (1.9) (39.2) Control118 23 14 81 0 0 0 0 Dox (68.6) No Dox 107 13 18 76 0 0 0 0 (71.0)

CMV, a ubiquitously active promoter, drives the pSF2. In all these setsof experiments, about half the injected and control fish were immersedin a solution of 150 ppm doxycycline (dox) to evaluate the efficiency ofrepression. The data were pooled from 3 separate sets of injections.

Following pSF2 injection and repression, the proportion of embryosexpressing EGFP in the dox treated group was much lower from that ofuntreated group (11% vs 59%). These results confirm Example 6 that theapplicant has achieved temporal control of genes under the regulation oftet responsive promoter in zebrafish.

However, as in case of pSF1, there was no correlation between theembryos expressing EGFP and those with a dorsalized deformity. Althoughthe CMV is a ubiquitously expressing promoter, the applicanthypothesized that the mosaic distribution of injected construct may haveprecluded consistent expression in the BMP2 expression domains.

The results from injection and repression of pSF3, in which the tTA isdriven by zebrafish SMAD5 promoter are presented in Table 8. TABLE 8Results of the repression experiment following injection of pSF3 andpSF2 constructs (100 ng/μl) in the zebrafish embryos. Numbers inparenthesis are percentages. Dead Dead Glow No Glow Treatment Total 5HPI 24 HPI # Live Deformed Normal Total Deformed Normal Total pSF3 NoDox 60 9 13 38 15  11  26 0 12 12 (63.3) (39.4) (28.9) (68.4) (31.5)(31.5) Dox 52 11 14 27 5 5 10 1 16 17 125 ppm (51.9) (18.5) (18.5)(37.0) (3)    (59.25) (62.9) Control No Dox 85 29 0 56 — — — 5 51 56(65.8) (8.9) (91.0) (100)    Dox 86 24 20 42 — — — 0 42 42 150 ppm(48.8) (100)    (100)    pSF2 No Dox 58 11 6 41 8 16  24 2 15 17 (70.6)(19.5) (39.0) (58.5) (4.8) (36.5) (41.4) Dox 58 18 14 26 1 2  3 9 14 23150 ppm (44.8)  (3.8)  (7.6) (11.5) (34.6)  (53.8) (88.4)

The applicant included pSF2 injections in this set of experiments aspositive controls for repression. Repression of embryos injected withpSF3 were carried out in rearing medium containing 125 ppm dox, unlikethe 150 ppm employed for pSF2 injected groups. This was because inpreliminary experiments the applicant encountered higher mortalityassociated with 150 ppm dox and pSF3 injected embryos (data not shown).

As for pSF2, treatment with dox reduced substantially the percentage ofsurviving embryos exhibiting EGFP expression and swirl-like deformies,confirming repression. Unlike the pSF2 construct, there was a clearassociation between. EGFP expression and a dorsalizied mutation, the twoco-expressing in close to 40% of the embryos surviving past 24 hpi. Thisconfirms that the SMAD5 promoter effectively expressed the BMP2double-stranded blocker, causing developmental arrest in un-repressedembryos. The applicant hypothesize that the increased efficiency ofSMAD5 promoter in the complete Sterile Feral Construct over that of BMP2promoter results from its potential early zygotic activation, ensuringthe transcription of blocker molecules much before expression of thenative BMP2 transcripts. Since the smad5 is known to be expressedmaternally (Hild et al., 1999), it is likely to function even moreeffectively in permanently transformed lines.

The applicant also built and tested a Sterile Feral Construct forzebrafish using mis-expression of the BMP2 gene as the blocker sequence(pSF4). As predicted, injection of pSF4 resulted in overexpression ofBMP2, resulting in fish with ventralizied mutations (FIG. 18A-C, arrow.Majority of the deformed fish co-expressed EGFP and in some instancesthe EGFP expression was closely associated with the ventralized tissue(FIG. 18C). As summarized in Table 9, the large majority of the EGFPexpressing embryos also had ventralized phenotypes as shown in FIG.18A-C. TABLE 9 Results of pSF4 injection (100 ng/μl) into zebrafishembryos Total No. Dead No No Glowing No. non-Glowing Treatment No. 5 HPI24 HPI Live Deformed Normal Deformed Normal PSF4 234 104 37 93 33 31 722 Injected (44.4) (15.8) (39.7) (35.4) (33.3) (7.5) (23.6) Control 118 46 10 68 — — 3 65 (4.4) (95.5)

EXAMPLE 8 Transfection of Pacific Oysters

Mature oysters (Crassostrea gigas) were obtained from local hatcheriesin Tasmania and New South Wales, and held in artificial seawater at 10°C. until required. Eggs were collected from 2-3 females by stripping thegonads and were pooled, rinsed on a 20 μm mesh, and left to condition inartificial sea water for 2 h. Sperm were stripped from male gonads,diluted to approximately 10,000 gametes/μl, and used immediately forelectroporation-mediated nucleic acid delivery. Plasmid DNA (50 μg/ml)or double-stranded RNA (dsRNA; 1 μg/ml) was delivered into 1×10⁶ spermusing a BioRad Gene Pulser II electroporator in 0.2 cm gapelectroporation cuvettes. Sperm were subjected to a singleelectroporation pulse (50 V, 100% modulation, 10 kHz, 12.5 msec) andimmediately mixed with 5000 oocytes. Fertilized embryos and developinglarvae were reared at 20° C. in artificial seawater containing 0.1 μg/mlchloramphenicol. Surviving larvae were counted after 24 h development.For experiments in which the Drosophila melanogaster heat shock promoterwas used to drive expression of the delivered genes, a 1 h heat shock at37° C. was provided either at 2 h or 18 h post fertilization, anddevelopment was then permitted to proceed at 20° C.

The applicant developed and tested transfection techniques for Pacificoyster eggs and larvae using genes encoding enhanced green fluorescentprotein (EGFP, Clontech), glucuronidase (GUS), and red fluorescentprotein (RFP, Clontech). Efficacy of electroporation as a transfectionmethod of oyster sperm, using EGFP as a reporter gene was tested. Twodifferent constructs, containing the EGFP gene under the control ofeither the CMV or Drosophila heat shock (Hsp) promoter were deliveredinto sperm using electroporation, and EGFP fluorescence was monitoredusing microscopy and fluorometric assays. Oyster embryos and larvaedisplayed a moderate level of autofluorescence that obscured detectionof low levels of EGFP. Consequently, it was seldom possible to visuallydistinguish transfectants from non-transfectants when the EGFP gene wasunder the control of the CMV promoter using the construct pBiT(CMV)-EGFP(SEQ ID NO:18) as compared to EGFP expression levels observed usingpBiT(dHSP)-EGFP (SEQ ID NO:19) following heat shock. However, EGFP andRFP were easily detected when expressed under the control of the D.melanogaster heat shock promoter, using constructs pBiT(dHSP)-EGFP (SEQID NO:19) and pBiT(dHSP)-RFP-oHoxDS/BH (SEQ ID NO:20) respectively. Byvisual inspection, it was estimated that approximately 60% of thesurviving trochophore larvae were transfected (Table 10). TABLE 10Genetic Electro- construct % larvae with EGFP fluorescence porationPromoter/ Heat EGFP relative applied Reporter shock fluorescence¹ tocontrols − − − 0 ± 0.2 1 ± 0.2 + − − 0 ± 0.2 1 ± 0.2 − CMV/EGFP − 1 ±0.5 1 ± 0.3 + CMV/EGFP − 5 ± 3   1.5 ± 0.2   − Hsp/EGFP + 4 ± 1   1.2 ±0.3   + Hsp/EGFP − 24 ± 10   2.4 ± 0.7   + Hsp/EGFP + 61 ± 15   14.3 ±1.1  ¹Larvae with EGFP fluorescence visibly greater than that seen innon-transfected controlsThe values represent the means and standard errors for three separateexperiments.

To quantitatively assess EGFP and RFP fluorescence, larvae werehomogenized in homogenization buffer (50 mM sodium phosphate, pH 7.0, 10mM EDTA, 0.1% Triton X-100, 0.1% Sarkosyl, 10 mM mercaptoethanol), andprotein extracts were measured for fluorescence using a BMG FLUOstarfluorometer.

Transfection efficiencies were also assessed in a second set ofexperiments that examined delivery of pHSP-GUS construct (SEQ ID NO:21).The pHSP-GUS construct was made in a two step fashion. First, the D.melanogaster heat shock promoter and terminator were isolated from thepCaSpeR-hs plasmid (Thummel and Pirrotta, 1992, Drosophila InformationService 71: 150) by PCR using the two primers: Dmhsp Forward Primer5′-GAATTCCTAGAATCCCAAAACAAACTGG-3′ SEQ ID NO: 31 Dmhst Reverse Primer5′- GGATCCTGACCGTCCATCGCAATAAAATGAGCC-3′ SEQ ID NO: 32

The resulting amplicon was cloned into the T-tailed vector pGEM-T-Easy(Promega) according to the manufacturer's directions to produce thepGEMhsp70 plasmid. The second step involved excision of the geneencoding the β-glucuronidase gene (gus) from the plasmid pBacPak8-GUS(Clontech) using the restriction endonucleases NcoI and EcoRI. The endsof the 1.8 kb gus fragment were then converted to blunt ends using theKlenow fragment of E. coli DNA polymerase. The pGEMhsp70 plasmid wasthen linearized at the polylinker site between the promoter andterminator sequences using BglII and the ends were converted to bluntends using the Klenow fragment. The 1.8 kb gus gene fragment was finallyligated into the blunt-end BglII site to produce the pHSP-GUS plasmid(SEQ ID NO:21). The pHSP-GUS construct expresses GUS under the controlof the D. melanogaster heat shock promoter (Table 11). TABLE 11 Efficacyof electroporation as a transfection method of oyster sperm, using GUSas a reporter gene. Genetic GUS construct activity Electro- Promoter/Heat % GUS relative to poration reporter gene shock survival activity¹controls − — none 100 ± 4  4.2 ± 0.3 1.0 + — none 95 ± 5 4.3 ± 0.3 1.0 −CMV/GUS none 93 ± 5 4.4 ± 0.4 1.0 + CMV/GUS none 92 ± 6 6.7 ± 0.8 1.6 −Hsp/GUS yes 92 ± 5 4.5 ± 0.5 1.1 + Hsp/GUS none 91 ± 6 10.5 ± 0.5  2.5 +Hsp/GUS yes 90 ± 4 83.2 ± 5.4  19.8¹GUS activity expressed as fluorescence units/μg protein.Values represent the mean and standard error for three separate spawningexperiments, each with three replicates.

GUS activity in these experiments was measured using a fluorometricassay as previously described (Jefferson, R A 1987).

Fluorometric assays of larval extracts confirmed that electroporation ofsperm could deliver foreign DNA into oyster embryos (Tables 10 and 11).In the absence of electroporation, little or no reporter gene expressionwas detected in transfected larvae. With electroporation, cleardifferences were observed in the relative strengths of the two differentgene promoters tested. Expression of the reporter genes wasapproximately 1.6 times higher using the heat shock promoter, even inthe absence of heat shock, compared to expression levels observed usingthe CMV promoter. With heat shock, reporter gene expression increasedanother 6-8 fold.

EXAMPLE 9 The Repressible Element in Oysters

Tet-Off™ control of EGFP expression was first assessed in oyster heartprimary cell culture, using culturing methods previously described (Mol.Marine Biol. Biotech. 5: 167-174). Oyster cells were first transfectedwith the pTet-Off plasmid (Clontech, Genbank ACC# U89929)., usingEffectene liposomes (Qiagen), and placed under neomycin selection for 2weeks. The cells were then co-transfected with the pBI-EGFP reportergene plasmid (Clontech #PT3146-5) and the selection plasmid pTK-Hyg(Clontech, GenBank Accession #: U40398). Dually transfected cells werethen treated with 1 μg/ml doxycycline and EGFP expression was assessed72 h later. Doxycycline was then removed from the medium, cells werewashed in PBS, and incubated for a further 96 h to determine if EGFPexpression had changed. It can be seen from Table 12 that a smallpercentage of cells were observed to express EGFP in the absence ofdoxycycline. TABLE 12 Tet-Off ™ Control of EGFP Expression in OysterCell Culture Transfection and doxycycline (Dox) % cells expressingTreatment EGFP None 0 pTet-Off 0 pBI-EGFP 0 pTet-Off + pBI-EGFP (no Dox)2.2 ± 0.4 pTet-Off + pBI-EGFP (+ Dox for 72 h) 0 pTet-Off + pBI-EGFP (+Dox for 72 h, 0.5 ± 0.2 followed by removal of Dox for 96 h)

The low double transfection rates are presumably due to most cellsacquiring the pTK-Hyg plasmid without acquiring the pBI-EGFP plasmid.Addition of doxycycline to the medium resulted in complete repression ofthe EGFP reporter gene expression. When the doxycycline was removed, thelevel of reporter gene expression increased after 96 h, indicating thatthe repression is reversible.

The results in Table 12 indicated that gene expression in oyster cellscan be regulated using the Tet-Off™ system, and hence similarexperiments were conducted in oyster larvae.

Oyster embryos were transfected with the pBiT(HSP)-EGFP plasmid (SEQ IDNO:19), which encodes the tetracycline (or doxycycline)-controlledtransactivator (tTA=Tet-Off™) under control of a heat shock promoter,and contains the EGFP reporter gene under the control of thetetracycline (doxycycline) response element (TRE). The constructpBiT(HSP)-EGFP (SEQ ID NO:19) was prepared as follows. Four fragmentswere prepared and ligated together to create the construct. The first,was obtained by digesting pHSP70-1MCS (SEQ ID NO:22) with XhoI and XbaIfollowed excision and gel purification of the appropriate XhoI/XbaIfragment containing the Drosophila HSP70 promoter. The second wasobtained by digesting pTet-Off (Genbank ACC# U89929) with XbaI andHindIII and gel purifying the appropriate fragment containing thetet-responsive transcriptional activator (tTA) and SV40 poly adenylationsignal. The third fragment was obtained by digesting pBI-EGFP (Clontech,PT3146-5) with HindIII and SapI and gel purifying the appropriatefragment containing the TRE and CMVmin bidrectional promoter andmultiple cloning site. The fourth fragment was obtained by digestingpTet-Off (Genbank ACC# U89929) with XhoI and SapI and gel purifying theappropriate fragment containing the vector backbone and ampicilinresistance gene.

The construct expresses the tet-responsive transcriptional activator(tTA) from the Drosophila HSP70 promoter (PHSP70) which in turnactivates expression of EGFP under control of the tetracycline-responseelement, or TRE. Oyster sperm were transfected with the construct usingelectroporation, and oocytes were fertilized and allowed to develop for24 hours in the presence or absence of 5 μg/μl doxycycline. In theabsence of doxycycline, EGFP was expressed in transfected oyster larvae,and when doxycycline was added, the EGFP expression levels dropped tolevels equal to that of non-transfected embryos (Table 13). The resultsfrom the tissue culture and embryo transfections indicate that transgeneexpression in oysters can be effectively controlled using the Tet-Off™system. TABLE 13 Regulation of EGFP Expression Using Doxycycline inOyster Larvae Transfected with pBiT(HSP)-EGFP (SEQ ID NO.19)Fluorescence (FU/μg protein) Total fluorescence (incl. Corrected forTreatment Regime autofluorescence) autofluorescence Non-transformed 320(±21) 0 (±21) control −Dox, − heat shock 426 (±24) 106 (±24)  −Dox, +heat shock 1025 (±78)  705 (±78)  +Dox, + heat shock 215 (±27) 0 (±27)Values represent the mean and standard deviation for two separatespawning experiments, each with three replicates.

EXAMPLE 10 Blocking Expression of a Developmental Gene in Oysters

The applicant has identified conserved gene functions which are crucialto larval development in oysters and characterised two suitablecandidate sequences as targets for antisense or dsRNA knockout.Disrupting this gene function is then lethal to the animal (larvae)because transcription factors are prevented from binding and initiatingcascades of gene activity required for morphogenesis (bodyconstruction). The applicant chose to target the DNA binding ability ofa class of transcription factors known as “Helix-loop-Helix” factorsthat bind DNA during the development of animal body plans (reviewed byStein et al., 1996; and also see de Rosa, 1999). The applicant isolatedtwo partial gene sequences comprising this crucial and highly conservedDNA binding sequence from a Pacific oyster cDNA library (HoxCg1 andHoxCg3; SEQ ID NOS.: 23 and 24, respectively). Alignments of thesequence of this evolutionary conserved class of genes and phylogeneticanalysis have revealed that this sequence is indeed a HOX gene and ispreviously undescribed in oysters (FIG. 19).

The applicant identified two oligonucleotide sequences that arecandidates for antisense larval pesticides. An oyster specificantisense:

-   5′-GAGATCGTTCAGTCAGCG-3′ SEQ ID NO:25.    and a broader spectrum antisense-   5′-CATGSGSSGGTTTTGGA 3′ SEQ ID NO:26.    wherein “S” represents the base guanine or cytosine. These sequences    are potentially capable of truncating vital gene products, and hence    preventing their function in vivo.

The applicant synthesized and tested antisense and double strandedblockers for the gus gene from Escherichia coli, Hox CG1 (SEQ ID NO:23),and Hox CG3 (SEQ ID NO:24). RNA was prepared by in vitro synthesis forthese three different genes or gene fragments: the 1.8 kb open readingframe of the gus gene from E. coli; the 129 bp fragment of oyster geneHox Cg1 (SEQ ID NO:23, AGAL ref# NM99/09101); and the 129 bp fragment ofthe oyster gene HoxCG3 (SEQ ID NO:24, AGAL Ref#NM99/09102). The DNAfragments were each cloned into pBluescript SK(+), the vectors werelinearized with either HindIII or PstI, and T3 or T7 RNA polymerase(Promega) was used to generate sense or antisense RNAs, respectivelyusing a commercially available in vitro transcription kit (Promega,Madison Wis.). The resulting samples were then digested with DNase I for15 minutes at 37° C. To produce double stranded RNA (dsRNA), equimolaramounts of the sense and antisense RNAs were mixed and heated to 80° C.and allowed to cool slowly to room temperature thus forming dsRNA. TheRNA was extracted with phenol/chloroform and then chloroform,precipitated with ethanol, and resuspended in 10 mM Tris-HCl, pH 9.Formation of dsRNA was confirmed by resolving the annealed andnon-annealed RNAs on a 1% agarose gel in TBE (90 mM Tris-borate, 2 mMEDTA, pH 8.0).

The in vitro transcribed dsRNAs, plus sense, and antisense RNAs for theGUS, HoxCG1 and HoxCG3 genes were delivered into oyster sperm byelectroporation using a set of conditions previously found to be optimalfor delivery of a reporter gene construct (dHSP70-GUS). Transfectionsfor the control treatments were carried out in RNA free sea water.Delivery of sense and antisense RNAs had no or only a small effect onthe number of individuals that developed, relative to the non-treatedcontrols (Table 14). TABLE 14 Effect on Early Larval Development ofOyster Transfected with In Vitro Transcribed Sense (S), antisense (AS),and double-stranded (DS) RNAs of three different gene sequences, GUS,HoxCG1, HoxCG3 RNA delivered % survivors at 24 h % arrested into spermdevelopment¹ development² control 100 ± 3  5 ± 1 GUS - (DS) 94 ± 5 7 ± 3HoxCG1 - (S) 91 ± 5 9 ± 4 HoxCG1 - (AS) 85 ± 9 17 ± 5  HoxCG1 - (DS) 71± 7 79 ± 10 HoxCG3 - (S) 92 ± 4 8 ± 4 HoxCG3 - (AS) 87 ± 6 15 ± 3 HoxCG3 - (DS) 79 ± 7 23 ± 5 ¹Percentage of embryos that developed into trochophores,relative to non-treated controls²Includes all individuals (embryos and larvae) thatfailed to develop to the D-hinge larval stage

Transfection with dsRNA for the GUS gene had no obvious effect ondevelopment, but transfection with dsRNAs specific to the HoxCG genesresulted in increased numbers of individuals showing arrested earlylarval development. The dsRNA specific to the HoxCG1 gene was the mosteffective dsRNA, with almost 80% of individuals failing to developbeyond the trochophore stage of larval development (Table 14).

Screening for mutant phenotypes in the resulting larvae revealed severedevelopmental mutants especially in the treatments containing dsRNA forboth gene constructs, but not the RNA-free controls (FIG. 20, Table 14).Fatal embryonic distortions due to the double stranded blocker of HoxCG1can be broadly classified as defects in the anterior/posterior axisformation including associated structures (such as the velum) and forHoxCG3 as defects in velum and body—perhaps premature velum release.

To test whether dsRNA could reduce expression of a gene in oyster cells,primary cell cultures were first transfected with the pHSP-GUS plasmid(SEQ ID NO:21). After two days of growth, the dsRNA specific to the gusgene was delivered into these cells by transfection using Effecteneliposomes (Qiagen). After 72 h, the level of GUS activity was measured.The cells transfected with the dsRNA showed a 76% reduction in thereporter gene activity compared to similarly aged gus-transfected cells(Table 15). TABLE 15 Reduced GUS Transgene Expression in Oyster CellsTransfected with In Vitro Transcribed dsRNA GUS Gene Expression (ρmol MUproduced/min) % decrease in No dsRNA added dsRNA added gene expression42 ± 13 10 ± 4 76

In vivo expression of dsRNA was achieved by transfecting oyster larvaewith the pBiT(dHSP)-RFP-oHoxDS/BH plasmid (FIG. 21; SEQ ID NO:20), whichcontains the heat inducible promoter (P_(HSP70)) from D. melanogasterdriving the expression of a hairpin RNA molecule specific to the HoxCG1gene. The construct was prepared as follows. SEQ ID NO:23 (AGAL ref #:NM99/09101) was used as a template to generate a PCR fragment using thefollowing primers: CG1.1.Sal.for Forward primer:5′-ATGGATGTCGACTCAGACGCTGGAG-3′ SEQ ID. NO.: 27 And CG1.1.Pst.revReverse primer: 5′-GATTCACTAGTCAATTCCTGCAGTT-3′ SEQ ID NO: 28This fragment was then cloned into the pCR®2.1-TOPO (Invitrogen) cloningvector. Two separate fragments, an EcoRI/EcoRI and a SalI/PstI, bothcontaining the HoxCG1.1 (SEQ ID NO:23), were digested out of thisconstruct for use in further ligations. The latter fragment (SalI/PstI)was inserted into the dsRNA(BMP2) construct (AGAL ref# NM99/09100) whichhad been digested with SalI and PstI to remove the inverted BMP2sequence. This intermediate construct was then digested with EcoRI andSpeI to produce a fragment containing both the a 510 bp fragment of thezBMP2 cDNA from sequence 301-810 in the published cDNA sequence (Lee etal., 1998) and the Hox CG1.1 (SEQ ID NO:23) fragment. This EcoRI/SpeIfragment and the EcoRI/EcoRI fragment containing HoxCG1.1 were thencombined into a ligation reaction with pHSP70-1MCS (SEQ ID NO:22,containing the Drosophila heat shock promoter dHSP70 and its polyadenylation signal) digested with EcoRI and XbaI, to producepHSP-oHoxDS/BH (SEQ ID NO:29). This latter construct uses the Drosophilaheat shock promoter to drive expression of an mRNA consisting of aninverted section of the HoxCG1.1 followed by a section of BMP2 cDNA insense orientation followed by a segment of the HoxCG1.1 fragment insense orientation followed by the poly adenalation signal of theDrosophila heat shock promoter.

Oyster sperm were transfected with the DNA using electroporation, andoocytes were fertilized and larvae allowed to develop for 96 hours.Embryos were heat shocked for one hour at 3 hours post fertilization toinduce transcription of the dsRNAs. Even without heat shock,approximately a third of the larvae failed to develop beyond thetrochophore larval stage, and died within a few days (Table 16). TABLE16 Arrested Development of Oyster Embryos Transfected withpHSP-oHoxDS/BH plasmid (SEQ ID NO: 29) % arrested development no heatshock with heat shock non-transfected 5 ± 1 4 ± 1 phsp-GUS 6 ± 2 8 ± 3pHSP-oHoxDS/BH 33 ± 9  67 ± 16

With heat shock, over 65% of the larvae failed to develop. Since alllarvae are not transfected by the electroporation procedure, it islikely that those individuals that developed normally were nottransfected with the genetic construct. Non-transfected oyster embryosand embryos transfected with a plasmid expressing dsRNA for the GUS geneshowed no obvious reduction in survivorship (Table 16).

EXAMPLE 10 Complete Sterile Feral Construct for Oysters

Two different plasmids were prepared that used Tet-Off™ to control thein vivo expression of dsRNAs specific to developmental genes. The first,pBiT(CMV)-EGFP-zfBMP(DS), (SEQ ID NO:30), was designed to express thereporter gene EGFP as well as dsRNA specific to the zebrafish BMP2 genein the absence of tetracycline or doxycycline. The construct wasprepared as follows:

An intermediate constuct was first engineered using three separatefragments. The first was an XhoI/HindIII fragment that was obtained bydigesting pTet-Off (Genbank ACC# U89929) with XhoI and HindIII and gelpurifying the appropriate fragment containing the CMV promoter,tet-responsive transcriptional activator (tTA), and SV40 polyadenylation signal. The second fragment was obtained by digestingpBI-EGFP (CLONTECH) with HindIII and SapI and gel purifying theappropriate fragment containing the TRE and CMVmin bidrectional promoterand multiple cloning site (MCS). The third fragment was obtained bydigesting pTet-Off (Genbank ACC# U89929) with XhoI and SapI and gelpurifying the appropriate fragment containing the vector backbone andampicilin resistance gene. These three fragments were ligated togetherto form the intermediate construct pBiT(CMV)-EGFP (SEQ ID NO:18). Afourth fragment, obtained by digesting Seq.ID#4 (dsRNA(BMP2), AGAL Ref#NM99/09100) with EcoRI and HindIII and gel purifying the appropriatefragment containing a 510 bp segment of the zBMP2 cDNA from sequence301-810 and the inverted 286 bp segment of the cDNA (Bases 307-592) ofthe published zebrafish BMP2 cDNA sequence (Lee et al., 1998). ThisEcoRI/HindIII fragment was then blunt ended with T4 DNA polymerase andligated into the unique PvuII site of the MCS of pBiT(CMV)-EGFP to formthe construct pBiT(CMV)-EGFP-zfBMP(DS) (SEQ ID NO:30). This constructexpresses the tet-responsive transcriptional activator (tTA) from thestrong immediate early promoter of cytomegalovirus (P_(CMV)). The tTAfunctions to drive gene expression via the tetracycline-responseelement, or TRE. In the absence of tetracyline or doxycyline both EGFPand the blocker gene (double stranded BMP2 mRNA, cloned into the MCS)are expressed.

Sperm were transfected with either pBiT(dHSP)-EGFP (SEQ ID NO:19) orpBiT(CMV)-EGFP-zfBMP(DS) DNA, (SEQ ID NO:30), oocytes were fertilized,and allowed to develop for 24 hours in the presence or absence of 5μg/μl doxycycline. Embryos transfected with the pBiT(dHSP)-EGFP DNA werenot heat shocked so that EGFP expression would be similar in bothtransfections. When oyster embryos were transfected with this construct,lower hatch rates and poorer larval survival rates than those ofnon-transfected controls were observed (Table 17). TABLE 17 Tet-Off ™Control of EGFP and dsRNA-zfBMP Expression in Oyster Embryos % survival(relative to EGFP (FU/μg Construct control) protein) injected −Dox +Dox−Dox +Dox Non-transfected 100 + 5  100 + 3   0 + 10  0 + 11pBiT(dHSP)-EGFP 77 + 6 95 + 3 31 + 8  0 + 8 pBiT(CMV)-EGFP- 71 + 8 92 +4 20 + 11 0 + 9 zfBMP(DS)

When doxycycline was added to the water, this trend was reversed. Mostof this arrested development however, may be caused by expression ofEGFP, as similar levels of arrested development were observed whenembryos were transfected with the pBiT(dHSP)-EGFP plasmid (withoutexposure to heat shock), and normal developmental rates were restoredwhen doxycycline was added to the water. It cannot be excluded however,that the zebrafish dsRNA has caused some small degree of developmentalarrest in the oysters, as the BMP2 may have an as yet unidentifiedorthologue with enough sequence identity to zfBMP2 to be affected bythis dsRNA molecule.

The second Sterile Feral Construct tested for oysters, expresses the tTAunder the Drosophila HSP. The tTA then drives expression of redfluorescent protein and double stranded oyster Hox via the TRE. Threeseparate fragments were ligated together to form this construct. Thefirst fragment was obtained by digestion of pBiT(dHSP)-EGFP, (Seq IDNO:19), with HindIII and NheI followed by gel purification of theappropriate fragment containing the Drosophila HSP promoter. The secondfragment was obtained by digesting pBiT(dHSP)-EGFP with NotI and MluIfollowed by gel purification of the appropriate fragment containing theTRE. The third fragment was obtained by digesting pHSP-oHoxDS/BH withMluI and. SpeI and gel purifying the appropriate fragment containing the510 bp fragment of the zBMP2 cDNA from sequence 301-810 in the publishedcDNA sequence (Lee et al., 1998). The fourth fragment was obtained byfirstly subcloning into pGEM3zf a KpnI/XbaI fragment containing thecoding region of red fluorescent protein (RFP) that was excised frompDsRed1-N1 (Clontech, PT3405-5) vector. The resulting plasmid was thensubjected to digestion with HindIII and PspOMI and the appropriatefragment containing the coding region of RFP was then gel purified fromthis reaction. This HindIII/PspOMI fragment was combined with theNheI/HindIII, NotI/MluI, and MluI/SpeI fragments to form the secondsterile feral oyster construct pBiT(dHSP)-RFP-oHoxDS/BH (SEQ ID NO:20;FIG. 21).

Sperm were transfected with the plasmid, oocytes were fertilized, andallowed to develop for 72 hours in the presence or absence of 5 μg/μldoxycycline. When oyster embryos were transfected with the secondrepressible sterile feral construct, a considerable percentage (67%)failed to develop beyond the trochophore stage of larval development andsubsequently died before reaching the D-hinge stage (Table 18). TABLE 18Reversible Arrested Oyster Larval Development Following Transfectionwith the Tetracycline-Responsive Plasmid phsp-BiT-RFP/dsRNA-HoxCG1Construct used for % arrested development transfection No doxycyclineWith doxycycline Non transfected 0 ± 5 0 ± 3 phsp-GUS 5 ± 3 4 ± 3pCMV-RFP 5 ± 2 4 ± 3 phsp-BiT-dsRNA- 67 ± 8  9 ± 4 HoxCG1/RFP

Addition of doxycycline to the water virtually prevented thedevelopmental arrest, and most embryos developed properly to the D-hingelarval stage, relative to the non-treated controls.

RFP expression was not easily detected by microscopy in embryostransfected with the RFP gene under the control of either a heat shockor a CMV promoter. A small amount of RFP was detected using fluorometricmeasurements of larvae transfected with the pCMV-RFP construct, butlittle RFP could be detected in larvae transfected with the repressibleanti-development construct (results not shown). As many of the embryostransfected with this latter construct fail to develop, the lack of RFPexpression is not surprising. Attempts to detect RFP in early and latestaged embryos were unsuccessful, using either RFP-expressing construct.

EXAMPLE 11 Development of a Repressibly Sterile Mouse

Development of the sterile feral construct for mice parallels thatdetailed above for zebrafish, and involves identification of a suitabletarget gene and associated promoter, engineering these into a constructwith the Tet On/Off repressible system, and then-testing, in this casein cell lines, prior to production of a transgenic mouse model for thesterile-feral concept.

There are many genes known to have adverse effects on fertilisation,development or reproduction in mice. These genes can be readilyidentified through literature and database searches (Medline, mouseknock out database, Genbank etc.). These candidate genes fall mainlyinto the category of genes that are required for specific developmentalprocesses during embryogenesis. Furthermore, genes that are involved instages of fertilisation and implantation are also potential candidategenes for this fertility control technology.

Developmental stages identified as potential sterile feral constructtargets are classified under one of the following general areas:fertilisation, preimplantation, post implantation (until neurulation)and organogenesis stages. The latter stages include factors such asthose associated with the specification of male and female reproductiveorgans (Cunha et al., 1976). Proteins involved in these stages may havedifferent roles such as morphogens, master genes, growth factors orreceptors.

Genes associated with fertilisation include such factors as proteinreceptors or ligands required for successful fertilisation.Preimplantation genes that can be manipulated to control their geneexpression and so achieve controllable fertility are also covered bythis patent and include genes encoding proteins such as growth factors,signaling molecules and their receptors.

The homeobox gene goosecoid is one of the first genes to be transcribedin the organizer region of the mouse at the onset of gastrulation andRNA transcripts first appear in the dorsal mesoderm of the late blastula(Blumberg et al., 1991). The goosecoid gene is also highly conservedamong different species (FIG. 22). During mouse embryogenesis,expression of the goosecoid gene takes place in two different phases. Inthe first phase of expression, goosecoid gene expression can be detectedin the organizer between 6.4 to 6.7 days (Blum et al., 1992) and in thesecond phase it is detected during organogenesis from 10.5 day onwards(Gaunt et al., 1993) and expressed in some parts of head, the limbs andthe ventrolateral body wall. The homozygous knockout mutation ofgoosecoid in the mouse leads to defects late in development of theembryos. In particular, null homozygous goosecoid embryos are born withnumerous developmental defects and die within 24 hours of birth(Rivera-Perez et al., 1995). The observed phenotype is in accordancewith late expression of goosecoid in normal embryos, and it has beenproposed that the lack of an earlier phenotype is due to functionalcompensation by other orthologous genes such as gsc2.

At the promoter level, molecular studies have demonstrated thatexpression of goosecoid in Xenopus is mediated by the combined effectsof two regions of the promoter, the distal element (DE) and the proximalelement (PE). The DE responds directly to dorsal mesoderm inducingsignals such as activin and Vg1 (members of the TGF-β super family),whereas the PE responds indirectly to wnt signaling (McKendry et al.,1998). Sequence comparison among different species shows that theseproximal and distal elements are conserved among different species andthere may be a common mechanism for its activation (Blum et al., 1992).It was proposed that the DE responds directly to mesoderm inducingsignals such as activin, whereas the PE responds indirectly to Wntsignaling (Laurent and Cho, 1999) (FIG. 23).

Studies involving the goosecoid promoter in mouse and other species haveshown that the promoter region carrying these two elements are adequatefor reporter gene activity studies. These two elements are generallylocated within 500 bp from the transcriptional start site.

The goosecoid gene, in the form of sterile feral constructs, can be usedto demonstrate how a developmentally active gene can be manipulated tomaintain its temporal and spatial gene specification under repressiblepromoter elements.

EXAMPLE 12 Cloning the Goosecoid Gene Promoter

The goosecoid promoter was amplified by PCR using BALB/c genomic DNA.Primers were designed from Mus musculus goosecoid homeobox gene,promoter sequence, of the Genbank accession number Y13151.

The primers were as follows: Forward Primer5′-GGAGACAGGCAGTCCCGGTAGATC-3′ SEQ ID NO: 33 Reverse Primer5′-TGGGAATTGTCCCACTCTCTGCTC-3′ SEQ ID NO: 34The PCR conditions were as follows:

-   95° C.×3 min, 72° C.×1 min (hotstart), 58° C.×1 min, 72° C.×1 min    for 1 cycle. Then 95° C.×45 sec, 58° C.×1 min, 72° C.×1 min for 28    cycles. The reaction was completed by incubating the reaction at 72°    C.×10 min and 25° C.×5 min). The PCR product for the goosecoid    promoter was ligated into pGEM-T-Easy cloning vector (Promega Cat #    A1360).

EXAMPLE 13 Selection and Construction of Reporter Plasmids for TestingPromoter Function

Reporter genes for promoter expression in mammals are available in twoforms. Firstly reporter genes can be used to determine location ofexpression of a gene product. Examples of such commercially availablereporters include the Enhanced Green Fluorescent Protein (EGFP) and RedFluorescent Protein (RFP). Alternatively, other reporter genes can beused to quantitate relative levels of expression and include fireflyluciferase (LUC+) modified for optimal expression in mammalian systems.The reporter genes EGFP and LUC+ were selected for use in testingsterile feral constructs based on the goosecoid promoter in the mouse.

pSFM 1: goosecoid promoter expressing enhanced green fluorescent protein(FIG. 24; SEQ ID 35). The goosecoid promoter produced by PCR and clonedinto pGEM-T-Easy (see above) was subcloned into the pEGFP-1 vector(Clontech Cat. # 6086-1) by digestion with EcoR1 and cloned into theEcoR1 site of the MCS of pEGFP-1. The orientation of the goosecoidpromoter was confirmed by both restriction enzyme mapping andsequencing.

pSFM 2: goosecoid cDNA in pTRE (FIG. 25; SEQ ID 36). A goosecoid cDNAequivalent was prepared from a goosecoid genomic DNA clone. Thegoosecoid DNA clone was prepared by PCR using BALB/c mouse genomic DNA.Primers were designed from the published sequence of goosecoid (GenbankAccession # M85271). The goosecoid gene coding region is comprised of 3exons. PCR primers were designed to produce each of the exonsindividually and were cloned into bacterial plasmid vectors usingstandard molecular biology techniques. The cDNA for goosecoid was thenreconstructed by tandemly ligating the individual exons together to forma new clone. The exons can also be joined in other orientations toencode for various combinations of dsRNA or antisense of the goosecoidRNA.

The Primers used were designed from the entire coding region of thegenomic DNA (Sequence locations referred to goosecoid Genbank AccessionNumber=M85271) and were:

Design of PCR primers to amplify goosecoid exons 1, 2, 3. exon 1 (bp296-650); exon 2 (bp 1159-1418); exon 3 (bp 1765-1920): SEQ ID NO: 37Exon 1 forward (bp 296-316) 5′-GGTTAAGCTTATGCCCGCCAGCATGTTCAGC-3′ SEQ IDNO: 38 Exon 1 reverse (bp 631-650)5′-GCGGGGCCCTCGTAGCCTGGGGGCGTCGGGACGCAG-3′ SEQ ID NO: 39 Exon 2 forward(bp 1165-1183) 5′-CGAGGGCCCCGGTTCTGTACT-3′ SEQ ID NO: 40 Exon 2 reverse(bp 1398-1418) 5′-TTTGAGCTCCACCTTCTCCTCCCGAAG-3′ SEQ ID NO: 41 Exon 3forward (bp 1765-1785) 5′-GTCTGGTTTAAGAACCGCCGA-3′ SEQ ID NO: 42 Exon 3reverse (bp 1900-1920 5′-GGAATTCTCAGCTGTCCGAGTCCAAATC-3′Three exons were amplified by PCR using the above primers and thefollowing conditions;

-   95° C.×2 min, 40° C.×30 sec, 72° C.×45 sec for 1 cycle. Then 95°    C.×30 sec, 40° C.×30 sec, 72° C.×45 sec for 30 cycles. The reaction    was stopped by incubation at 72° C.×10 min and 25° C.×5 min.

Goosecoid exon 1-3 PCR products were cloned into Promega (Cat # A1360)pGEM-T-Easy cloning vectors. These clones were named pME 1, pME 2 andpME 3 for exon 1-3 in pGem-T-Easy respectively.

The strategy for producing the equivalent clone for the completegoosecoid cDNA coding region was as follows:

-   -   pME 2 was cut with ApaI and religated, to remove the EcoR1 site.        Pfu polymerase PCR of clone pME 3 was undertaken using the        primers and conditions for exon 3 as described above. This        generated a blunt-ended fragment which was then digested with        EcoRI. Following religation of pME2 (see step 1 above) with        EcoIcR1. Ligated together pME2 from (3) and digested PCR product        from (2) to produce pME 4.

Cut pME 1 with HindIII and then partial digest with ApaI (band size 370bp, external ApaI site).

Cut pME 4 with ApaI, followed by EcoR1.

Cut pBluescript SK− with HindIII followed by EcoRI Ligated (7) abovewith pME 4 product and pME 1 product to produce the complete goosecoidcDNA coding region. This clone was confirmed by sequencing anddesignated pCMH142 (SEQ ID 43).

pSFM 6: Goosecoid promoter expressing goosecoid cDNA fused to redfluorescent protein (FIG. 26). A 0.9 kb PCR fragment containing the fullcoding sequence of mouse goosecoid was amplified from pCMH142 using twoPCR primers: gsc F4 - 5′-TTAAGCTTGCCACCATGCCCGCCAGCATGT-3′ SEQ ID 44 gscR4 - 5′-TTGGATCCGCGCTGTCCGAGTCCAAATC-3′ SEQ ID 45These primers produced a goosecoid-containing fragment where the TGAstop codon was replaced with an alanine codon. The PCR primers were alsoused to introduce a HindIII site upstream of the ATG start codon and aBamHI site downstream of the alanine codon. This fragment was restrictedwith HindIII and BamHI and then inserted into the plasmid pDSRed1-N1(Clontech 6921-1) cut with HindIII and BamHI in order to generate pSFM 6(SEQ ID 46).

pSFM 7: Mouse goosecoid promoter expressing the tetracyclinetransactivator protein tTA (FIG. 27). SEQ ID 47.

The goosecoid tetracycline dependent transactivator plasmid wasconstructed by replacing EGFP of pSFM 1 with the 1008 bp coding regionregion (Genbank accession # U89930 bp 774-1781) of the tet-responsivetranscriptional activator (tTA) from the pTET-OFF plasmid (Clontech, Cat# K1620-A). The tTA coding region was amplified by PCR using Pfupolymerase, restricted by Age1 and EcoR1 and cloned into pSFM 1 toproduce pSFM 7.

pSFM 20: goosecoid promoter expressing luciferase+ protein (FIG. 28).SEQ ID 48.

A 0.7 kb (NotI end-filled with Klenow+BamHI) fragment coding for greenfluorescent protein region from pSFM1 was replaced with 1.6 kb (XbaI endfilled with Klenow enzyme+BamHI) luciferase+ coding fragment derivedfrom pXP1-G (Promega E1751).

pSFM 21: Promoterless luciferase+ (FIG. 29). SEQ ID 49.

A 1.6 kb luciferase coding EcoRI fragment was deleted from pSFM 20.

pSFM 23: pCMV promoter expressing luciferase+ (FIG. 30). SEQ ID 50.

A 1.6 kb (SacI+StuI) luciferase+ coding fragment of pSFM 20 was clonedinto pEGFP-N1 (Clontech 6085-1) cut with SacI+StuI.

pSFM 24: Equivalent to the tet-responsive enhanced green fluorescentprotein expression vector pTRE-EGFP (Clontech 6241-1)(FIG. 31) SEQ ID51.

pSFM 25: Tet-responsive expression vector pTRE-luciferase+ (FIG. 32).SEQ ID 52.

A 0.77 kb SalI+XbaI EGFP containing fragment of pSFM 24 was replaced bya 1.7 kb SalI+XbaI luciferase+ containing fragment derived from pXP1-G(Promega).

EXAMPLE 14 Selection of Mammalian Cell Lines

Mouse goosecoid was selected to demonstrate whether a developmental genecan be tightly regulated in the form of sterile feral constructs inmammalian cell lines. Most of the mainpulations using sterile feralconstructs based on goosecoid were therefore carried out in the mouseembryo cell lines P19 teratocarcinoma since it has been shown previouslythat the mouse goosecoid gene product is constitutively expressed in P19teratocarcinoma cell lines. NIH/3T3 cells (in which goosecoid geneexpression is absent) were used as controls.

In addition goosecoid reporter constructs were tested in non-transformedmouse primary embryonic fibroblasts. These cells display monolayered,anchorage dependent and contact inhibited growth in tissue culture.Using transient transfection with reporter and other plasmid constructs(reporters and blockers) the observed effects on these plasmids isexpected to reflect the anticipated effect in the whole organism.

Chromatin structure surrounding the inserted gene is also likely affectthe pattern of regulation of gene expression and so the choice of stablecell lines for gene expression is essential. For example, it is knownthat transfected DNA does not display the same accessibility totranscriptional factors as chromosomal DNA (Archer et al., 1992).Another important factor to consider is that the goosecoid promotercontains only 1.1 kb upstream to the transcription start site leading topotential restriction of access by nuclear and other transcriptionalfactors by surrounding DNA sequences and chromatin structure.

All cell lines were obtained from American Type Culture Collectionunless otherwise stated. These are P19 teratocarcinoma cells (ATCCnumber CRL-1825) and NIH/3T3 cells (ATCC number CRL-1658).

For transient transfection assays, P19 cells were cultured ongelatinized dishes in DMEM supplemented with 10% fetal bovine serum.Cells (0.3 million per well in 6-well cluster plates) were transfectedwith 5 μg reporter plasmid using transfection reagent ‘Geneporter’ fromGene Therapy Systems according manufacturer's recommendation.

Stably integrated P19 clones were obtained by using BioRad Gene PulserII electroporation system. 30 μg DNA electroporated into 10 millioncells under following conditions 960 μF and 0.16 kV in a 0.4 cm cuvette(0.4 kV/cm). The next day normal media were replaced with appropriateselection media (300 μg/ml G418).

Reverse transcriptase polymerase chain reaction (RT-PCR) was used toconfirm that the goosecoid gene is actively expressed in P19 cell lineswith the goosecoid specific primers exon 2 forward (SEQ. ID 39) and exon3 reverse (SEQ ID 42):

RT-PCR

cDNA was synthesized in a 50 μl reaction using 100 ng of poly(A) RNAextracted from various tissues and cell lines. The RNA was heated with amixture of random 6 base pair and oligo(dT) primers for 5 min at 65° C.and cooled to room temperature for 10 min. Reverse transcription wasperformed at 37° C. for 1 h after adding 5 μl. 10×RT buffer (Promega),20 U RNase inhibitor (Promega), 2 μl of 0.1 mM dNTPs and 50 U MMLVreverse transcriptase. The cDNA mixture was then heated for 5 min at 90°C. and stored at −20° C. until needed.

RT-PCR was conducted using 2 μl of cDNA in a 50 μl final reaction usinggoosecoid specific primers (FIG. 33). By comparison, RT-PCRamplification on NIH/3T3 cells gave negative results for goosecoid. Inboth cells, RT-PCR of a general housekeeping gene GADPH gave positivebands. In addition GFP expression from P19 cells containing the reporterplasmid pSFM 1 stably integrated was unaffected by repeated passaging orfreezing and thawing.

In order to measure the activity of the goosecoid gene, a cell culturesystem was developed that responds to tetracycline repression andpermits the measurement of gene activity using both fluorescencereporters.

Fluorescent and transmitted light images were acquired using a CCDcamera with a microscope. Fluorescence filter sets had an excitationwavelength of 480 nm, dichroic cut-on filter at 505 nM and an emissionfilters at 535 nM and 605 nM. The luminescence assays were conducted byusing a dark 96 well plate was done by Victor2 from Wallac or byTopcount NXT from Can berra Packard.

P19 cells were transiently transfected in 6 well plates with pSFM 20(goosecoid promoter-luciferase), pSFM 21 (promoterless luciferase) andpSFM 23 (CMV promoter-luciferase) using Gene Porter. Cells wereharvested at various times post-tranfection and assayed for luciferaseactivity using a Promega kit (Cat. # E1501) in a Top Count NXTluminometer.

Table 19 shows the luciferase activities of promoter reporter constructsshown in counts per second (cps) of transiently transfected in P19cells. TABLE 19 Hours pSFM 21 pSFM 23 pSFM20 24 254 78778 1263 48 604145403 3707 72 252 49936 1692

Maximum luciferase activity was observed 48 hours post-transfection forall plasmids. Luciferase activity from the goosecoid promoter construct(pSFM 20) was 6 fold higher compared to the promoterless construct (pSFM21). CMV driven luciferase activity (pSFM 23) was 200-300 fold higherthan for the promoterless luciferase (pSFM 21). Therefore 48 hours posttransformation was selected for optimal detection of luciferaseexpression.

Selection of a P19 cell line stably integrated with agoosecoid-dependent TET-OFF transactivator P19 cells were electroporatedwith pSFM 7 (Goosecoid promoter-TET/OFF) linearised with ApaLI andselected for stable integration.

Table 20 showes the luciferase activities of pSFM 25 (TRE luciferase+)shown in counts per second (cps) of transiently transfected in P19-pSFM7 cells. TABLE 20 pSFM 25 pSFM 25 Clone number without doxycycline withdoxycycline 9 582529 54858 12 417268 4396 29 616604 48260 32 27288819548 46 703470 8013

From 100 clones, one clone (46) was selected which demonstrated thehighest luciferase activity when transiently transfected with thereporter plasmid pSFM 25 (TRE-luciferase+). Addition of doxycycline at 1μg/ml reduced luciferase activity from pSFM 25 in this clone by 90 fold.This clone, containing stably integrated pSFM 7 was therefore designatedP19-pSFM 7 and used for further testing.

Reporter plasmids pSFM 20 (goosecoid promoter luciferase+), pSFM 21(promoterless luciferase+), pSFM 23 (CMV promoter luciferase+) and pSFM25 (TRE luciferase+) were transiently transfected into either P19 orP19-pSFM 7 (Goosecoid TET/OFF) cells to test the effectiveness of theTET-OFF genetic switch driven by goosecoid promoter. Table 21 shows theluciferase activities of transient transfection of reporter plasmids inP19 and P19-pSFM 7 cell lines. TABLE 21 Pl9-pSFM 7 cells P19 cellsPlasmids Average Fold Average Fold pSFM 20 365 6 610 5 pSFM 21 60 1 1211 pSFM 23 16031 267 44491 367 pSFM 25 368 6 183 1.5

P19-pSFM 7 but not the P19 cells show a 6 fold increase in luciferase+reporter activity when transfected with pSFM 25 compared to thepromoterless plasmid pSFM 21. This increase is comparable to theincrease seen when the cells are transfected with plasmids containingthe luciferase driven by the goosecoid promoter (pSFM 20). Therefore theP19-pSFM 7 cell line can be used to drive expression through pTREplasmids to the same level as plasmids driving expression from thegoosecoid promoter directly.

EXAMPLE 15 Construction and Testing of Blocker Plasmids

Antisense and double stranded blockers specific for goosecoid wereconstructed.

pSFM 5: Tet-responsive expression vector pTRE-goosecoid double strandRNA (FIG. 34). SEQ ID 57.

pSFM5 was derived from pSFM 2 and pSFM 9. A 0.48 kb PstI+BamHI fragmentof pSFM 9 was inserted into a 3.9 kb PstI partial+BamHI fragment of pSFM2 to produce pSFM 5.

pSFM 8: pCMV promoter expressing goosecoid antisense RNA (FIG. 35). SEQID 58.

A 0.8 kb EcoRI+KpnI fragment of pSFM 9 containing the goosecoid cDNA wasinserted into pdsRED-N1 (Clontech 6921-1) cut with KpnI+EcoR1. Thisclone was then cut with SmaI+HpaI to remove the RFP and religated toproduce pSFM 8.

pSFM 9: Tet-responsive expression vector pTRE-goosecoid antisense RNA(FIG. 36). SEQ ID 59.

A 0.78 kb HindIII Klenow end-filled+EcoRI fragment of pCMH142 was clonedinto pTRE cut with BamHI end-filled with Klenow+EcoRI.

The first stage for testing blocker constructs is to set up anappropriate cell system to detect expression of reporter constructs.Initially, either pdsRED-N1 (CMV promoter RFP), pSFM 6 (CMV promotergoosecoid cDNA fused to RFP) or pSFM 24 (TRE EGFP) were transfected intoP19-pSFM 7 cells to test the expression patterns of the EGFP, RFP andgoosecoid-fused to RFP proteins (FIG. 37). These tests show that RFP isexpressed in the cytoplasm when driven from a CMV promoter (FIG. 37,B).When goosecoid is fused to RFP and driven from a CMV promoter however,the RFP signal is now detected in the nucleus (FIG. 37C,D), whereas theEGFP is expressed in the cytoplasm of the same cells when expressedthrough the TRE promoter (FIG. 37D). This shows therefore, thatgoosecoid is efficiently transferred to the nucleus when fused to thereporter gene RFP and that this system can be used to testco-transfected blocker plasmids against goosecoid. In these cases, RFPexpression fused to goosecid in the nucleus is expected to be inhibitedin the presence of an appropriate blocker.

In order to assess various antisense and dsRNA blockers, pSFM 6 (CMVpromoter goosecoid fused to RFP) was transiently cotransfected into theP19-pSFM 7 (Goosecoid promoter TET/OFF) cells along with either pSFM 5(TRE promoter dsRNA goosecoid), pSFM 8, (CMV promoter antisensegoosecoid), pSFM 9 (TRE promoter antisense goosecoid) or pSFM 24 (TREpromoter EGFP). In these cases, significant difference could not bedetected between the various treatments in either the intensity ornumber of cells expressing RFP in the nucleus. There are severalpotential reasons for the absence of RNA blocker effects. First,antisense and dsRNA blockers may not be expressed at levels high enoughto effectively interfere with the target mRNA molecules. Secondly, theremay be cellular mechanisms in mammals that recognize and interfere withsuch constructs. Thirdly, the RNA inhibitory molecules may not be ableto access and block the RNA target.

The goosecoid gene, in the form of sterile feral constructs, was testedin mammalian cells to demonstrate whether plasmids DNA coding for SFblockers have effect any effect on blocking goosecoid expression. Wehave demonstrated the methods for producing stably integrated cell linesand the testing of blocker constructs based on goosecoid dsRNA andgoosecoid anti-sense. Our results suggest that post-transcriptionalsilencing through double strand RNA is unlikely to be very effective inmice. We therefore conclude that either the system described here isinsufficiently sensitive to detect RNA interference using the currentblockers or that these inhibitors are relatively ineffective in the P19mammalian cell line. Nevertheless, small effects in cell culture cantranslate into severe phenotypic abnormality when introduced into mice.

By contrast, over-expression and mis-expression of genes leading todevelopmental abnormalities has been demonstrated in mice (Zwijsen etal., 1999; Goodrich et al., 1999). It can be reasonably expectedtherefore that sterile feral blockers that cause over-expression ormis-expression of developmental genes through at tetracyclinerepressible system will succeed. However, sense constructs cannot beeasily tested using reporter systems. It is necessary to stablyintroduce such constructs into embryonic stem (ES) cells and producetransgenic mice to evaluate the extent to which development can bedisrupted.

EXAMPLE 16 Production of Transgenic Mice Using the Goosecoid Promoter

By using the goosecoid gene promoter (or similar) to drive expression ofknown proteins critical to early embryogenesis a transgenic mouse can bemade. Candidate sense blockers for expression from the goosecoidpromoter are gene products that are critical for development in themouse and are also normally expressed in the embryo during gastrulationat the same time as the goosecoid gene product. Two other proteins,Chordin and Noggin, are known to expressed within the same embryonicregion at times and locations similar to that of goosecoid (Bachiller etal., 2000). In particular, Chordin is expressed in the same region asgoosecoid at embryonic stage TS 11 in the primitive streak and node.

Double knock-out mice for Chordin and Noggin have been produced andthese show severe phenotypic defects in the prosenchephalon. Both ofthese proteins are therefore essential for successful development in themouse. These two genes are antagonisers of another gene product, BMP-4,which is expressed in the region adjacent to the primitive streak.Together, these three gene products contribute to the anterior/posteriorstructural features of the developing mice. Therefore, misexpression ofBMP-4 using the goosecoid promoter, within the primitive streak, whereNoggin and Chordal are expressed, will interefere with the balancebetween these gene products and be expected to produce a phenotype thatwill match the double knock-out for Chordin and Noggin. Many otherdevelopmental genes, particularly those involved with earlyembryogenesis could be misexpressed in a similar manner.

The following process can be used to generate a transgenic mouse lineexpressing repressible developmentally regulated blockers. Genetargeting in mice is regularly achieved using two different methods. Oneis by oocyte injection and the other is through gene insertion intoembryonic stem cells. The embryonic stem cell method is the mostpreferred for manipulations using the goosecoid gene since this gene isusually activated following removal of leukemic inhibitory factor (afactor used to maintain the cells in undifferentiated state) from theculture medium (Savatier et al., 1996). Testing for effectiveness ofreversible blockers on goosecoid expression in cell cuture can thereforebe tested in embryonic stem cells before being transferred into mousebut not in system using directly injected oocytes.

The manipulations and production of repressibly steile transgenic miceis readily achievable to those practiced in the art (Hogan et al.,1994). This involves the following steps:

Transfection, stable integration and selection of embryonic stem cellswith a sterile feral construct consisting of the goosecoid promoterdriving expression of tTA (Tet-Off) such as pSFM 7 (SEQ ID NO:48).

Transfection, stable integration and selection of the teracycinedependent effector construct consisting of the TRE (Tet-reponsivepromoter) driving expression of one of the following: goosecoidantisense or dsRNA in constructs such as pSFM 9 (SEQ ID NO:59) and pSFM5 (SEQ ID NO:57) or the cDNA for genes essential for development in theembryo around the time of primitive streak formation (such as BMP-4).

Conclusions

One type of “sterile feral” construct encompassed by the presentinvention consists of three components, a developmental or constitutivepromoter, a gene blocker sequence, and a repressible promoter fromClontech™'s commercially available Tet-Off system. The developmental orconstitutive promoter functions to drive expression of Tet-Off represserprotein (tTA, Clontech™) which binds to the tet responsive element(TRE-CMV_(min), Clontech™) that in turn drives expression of the geneblocker sequence. Expression of the blocker DNA sequence results inproduction of either antisense or double stranded mRNA to ultimatelyknock-out function of the target gene or mis-expression of a sensesequence, that causes distorted development and embryo death. Correctfunction of the sterile feral construct requires that functions of boththe developmental promoter and the target gene are confined to eitheroogenesis or embryogenesis. This can be achieved optimally by using astage-specific promoter, though it can also be achieved through use of adevelopmental blocker who's effects are also spatio-temporally confinedto early embryogenesis. Repression of the blocker sequence function isaccomplished through exposure to tetracyline which prevents the bindingof the tTA to the TRE-CMV_(min).

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1. A method of controlling fertility in an animal comprising the stepsof: 1) stably transforming an animal cell or single celled embryo with aconstruct comprising: a) a first nucleic acid molecule, which isactivated in a defined spatio-temporal pattern, and which is operablylinked to b) a second nucleic acid molecule, which encodes atransactivating protein; and c) a third nucleic acid molecule, which isoperably linked to a fourth nucleic acid molecule,  wherein activationof said first nucleic acid molecule controls the expression of thesecond nucleic acid molecule, which in turn activates the third nucleicacid molecule, which effects the expression of the fourth nucleic acidmolecule which encodes a blocker molecule which disrupts gametogenesisor embryogenesis in the animal; and 2) and growing a whole animaldirectly from that cell or implanting the cell into a host animal,whereby a whole animal develops from the implanted cell.
 2. The methodof claim 1, wherein said first or said fourth nucleic acid molecule istransiently activated or transiently affects development in a definedspatio-temporal pattern.
 3. The method of claim 1, wherein each of thefirst, second, third and fourth nucleic acids is genomic DNA, cDNA, RNA,or a hybrid molecule thereof.
 4. The method of claim 3, wherein thenucleic acid molecule is a full-length molecule, or a biologicallyactive fragment thereof.
 5. The method of claim 1, wherein the firstnucleic acid molecule is a DNA molecule encoding a promoter region. 6.The method of claim 5, wherein the promoter is activated only duringembryonic development and/or gametogenesis, and is crucial forcompletion of embryogenic development and/or gametogenesis.
 7. Themethod of claim 5, wherein the promoter comprises the nucleotidesequence of SEQ ID NO:1, SEQ. ID NO:8, or SEQ ID NO:60.
 8. The method ofclaim 1, wherein the second nucleic acid molecule is a cDNA moleculeencoding a tetracycline-responsive transcriptional activator protein. 9.The method of claim 8, wherein said tetracycline-responsivetranscriptional activator protein comprises the nucleotide sequence ofSEQ ID NO:2.
 10. The method of claim 1, wherein the third nucleic acidmolecule encodes a repressible promoter.
 11. The method of claim 10,wherein the promoter consists of a tet-responsive element (TRE) which iscoupled to and tightly regulates a minimal promoter region.
 12. Themethod of claim 11, wherein said minimal promoter region is a PminCMVpromoter region that comprises the sequence of SEQ ID NO:3.
 13. Themethod of claim 1, wherein the fourth nucleic acid molecule encodes ablocker molecule selected from the group consisting of an antisense RNA,a double-stranded RNA (dsRNA), a sense RNA and a ribozyme.
 14. Themethod of claim 13, wherein the molecule is dsRNA or sense RNA that whenmis-expressed disrupts development in a defined spatio-temporal pattern.15. The method of claim 13, wherein the RNA is encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO:13, SEQ IDNO:62, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID:61.
 16. The method ofclaim 1, wherein said animal cell or said single celled embryo istransformed with said construct by microinjection, transfection orinfection, wherein said construct stably integrates into the genome ofsaid cell or said single celled embryo by homologous recombination. 17.A nucleic acid molecule, which encodes a promoter and is transientlyactivated in a defined spatio-temporal pattern, wherein said nucleicacid molecule comprises the nucleotide sequence of SEQ ID NO:1, SEQ IDNO:8, or SEQ ID NO:60.
 18. A nucleic acid molecule, which encodes apromoter having: a) a nucleotide sequence as shown in SEQ ID NO:1, SEQID NO:8 and SEQ ID NO:60; b) a biologically active fragment of thesequence in a); c) a nucleic acid molecule which has at least 85%sequence homology to the sequence in a) or b); or d) a nucleic acidmolecule which is capable of hybridizing to the sequence in a) or b)under stringent conditions.
 19. A nucleic acid molecule that encodes thecoding region of a gene including: a) a nucleotide sequence selectedfrom the group consisting of SEQ ID NO:63, SEQ ID NO:23, SEQ ID NO:24and SEQ ID NO 61; b) a biologically active fragment of any one of thesequences in a); c) a nucleic acid molecule that has at least 85%sequence homology with any one of the sequences disclosed in a) or b);or d) a nucleic acid molecule that specifically hybridizes to any one ofthe sequences disclosed in a) or b) under stringent conditions.
 20. Anucleic acid molecule that encodes a blocker molecule that disruptsgametogenesis or embryogenesis in an animal, wherein the blockermolecule is encoded, or partially encoded, by a sequence selected fromthe group consisting of SEQ ID NO:13, SEQ ID NO:62, SEQ ID NO:23 and SEQID NO:61.
 21. The nucleic acid molecule of claim 20, wherein the blockermolecule is selected from the group consisting of an antisense RNA, adsRNA, a sense RNA and a ribozyme.
 22. The nucleic acid molecule ofclaim 21, wherein the molecule is a dsRNA or a sense RNA that whenmis-expressed disrupts development in a defined spatio-temporal pattern.23. A transgenic non-human animal stably transformed with the nucleicacid molecule of claim
 17. 24. The transgenic non-human animal of claim23, wherein the animal is selected from the group consisting of fish,mammals, amphibians, and molluscs.